Use of lipid conjugates in the treatment of diseases

ABSTRACT

This invention provides compounds represented by the structure of the general formula (A):  
                 
wherein L is a lipid or a phospholipid, Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol, Y is either nothing or a spacer group ranging in length from 2 to 30 atoms, X is a physiologically acceptable monomer, dimer, oligomer, or polymer, wherein X is a glycosaminoglycan; and n is a number from 1 to 1000, wherein any bond between L, Z, Y and X is either an amide or an esteric bond.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 09/756,765, filed Jan. 10, 2001, which claimspriority from a Provisional Application U.S. Ser. No. 60/174,907, filedJan. 10, 2000, which is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention provides compounds represented by the structure of thegeneral formula (A):

wherein L is a lipid or a phospholipid, Z is either nothing,ethanolamine, serine, inositol, choline, or glycerol, Y is eithernothing or a spacer group ranging in length from 2 to 30 atoms, X is aphysiologically acceptable monomer, dimer, oligomer, or polymer, whereinX is a glycosaminoglycan; and n is a number from 1 to 1000, wherein anybond between L, Z, Y and X is either an amide or an esteric bond.

BACKGROUND OF THE INVENTION

Lipid-conjugates having a pharmacological activity of inhibiting theenzyme phospholipase A2 (PLA2, EC 3.1.1.4) are known in the prior art.Phospholipase A2 catalyzes the breakdown of phospholipids at the sn-2position to produce a fatty acid and a lysophospholipid. The activity ofthis enzyme has been correlated with various cell functions,particularly with the production of lipid mediators such as eicosanoidproduction (prostaglandins, thromboxanes and leukotrienes), plateletactivating factor and lysophospholipids. Since their inception,lipid-conjugates have been subjected to intensive laboratoryinvestigation in order to obtain a wider scope of protection of cellsand organisms from injurious agents and pathogenic processes.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a compound represented by thestructure of the general formula (A):

wherein

-   L is a lipid or a phospholipid;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein X is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between L, Z, Y and X is either an amide or an    esteric bond.

In another embodiment, the invention provides a process for thepreparation of a compound represented by the structure of the generalformula (A):

wherein

-   L is a lipid or a phospholipid;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein X is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between L, Z, Y and X is either an amide or an    esteric bond, comprising the steps of:    -   conjugating L to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, L is conjugated directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, L is conjugated directly to X,    -   thereby preparing a compound represented by the structure of the        general formula (A).

This invention provides lipid conjugates, comprising glycerol-derivedlipids including phospholipids, such as phosphatidylethanolamine, andphosphatidylserine, which when appropriately prepared by conjugation toa physiologically compatible monomer, dimer, oligomer or polymericmoiety, display an unexpected wide range and potency of pharmacologicalactivities. Administration of these compounds comprises effectivetreatment of a subject afflicted with diseases involving the productionof lipid mediators and/or impairment of glycosaminoglycan (GAG)functioning The diseases include disorder of smooth muscle cellproliferation, ischemic/reperfusion injury, obstructive respiratorydisease, airway and lung injury, colitis, Croln's disease, intestinalmucosal injury, central nervous system insult, multiple sclerosis, skindiseases, contact dennatitis, seboreic dermatitis, psoriasis,conjunctivitis, cardiovascular disease, including prophylaxis forinvasive procedures, atherosclerosis, invasive cellular proliferativedisorders, aterial stenosis and restenosis, primary cancer, metastaticcancer, hemolytic syndromes, sepsis, acute respiratory distresssyndrome, tissue transplant rejection syndromes, autoimmune disease,arthritis, viral infection, HIV infection, chlamydia infection, orhypersensitivity conjunctivitis.

In one embodiment, this invention provides administration of thesecompounds for the treatment of diseases which requires controllingphospholipase A2 activities, controlling the production and/or action oflipid mediators, amelioration of damage to cell surface byglycosaminoglycans (GAG) and proteoglycans, controlling the productionof oxygen radicals and nitric oxide, protection of lipoproteins fromdamaging agents, anti-oxidant therapy; anti-endotoxin therapy;controlling of cytokine, chemokine and interleukine production;controlling the proliferation of cells, controlling of angiogenesis andorgan vascularization; inhibition of invasion-promoting enzymes,controlling of cell invasion, controlling of white cell activation,adhesion and extravasation, amelioration of ischemia/reperfusion injury,inhibition of lymphocyte activation, controlling of blood vessel andairway contraction, protection of blood brain barrier, controlling ofneurotransmitter production and action or extracorporeal tissuepreservation.

In one embodiment, this invention provides phospholipase A2 inhibitors,thus controlling the production and/or action of adverse lipidmediators.

Additional mechanism by which these compounds ameliorate diseases, istheir functioning like cell surface glycosaminoglycans (GAG). Theconjugated moity anchored to the cell membrane by the lipid moity,mimicks the cell surface GAG and pioteoglycans in protecting the cellfrom damaging agents.

In another embodiment of the invention, new compounds are provided,representing low molecular weight lipid-conjugates, in particular lipidsbound through their polar head group to a mono- or disaccharide, acarboxydisaccharide, a mono- or dicarboxylic acid, a salicylate, anamino acid, a dipeptide, an oligopeptide, a bile acid, a fatty acid,cholesterylhemisuccinate, a trisaccharide, or a di- or trisaccharideunit monomer of a polyglycosaminoglycan, including repeating units ofheparin, heparan sulfate, hyaluronic acid, dextran, chondroitin,chondroitin-4-sulfate, chondroitin-6-sulfate, keratin, keratan sulfate,dermatin, and dermatan sulfate. These new compounds, as representativeof the class of lipid or lipid-conjugates of low molecular weight,exhibit the same wide range and potency of pharmaceutical activitiesmanifested by the higher molecular weight lipid-conjugates describedherein. Introduction of these novel compounds here expands the range ofuseful lipid-conjugatesas novel therapeutic drugs in the treatment ofspecific diseases.

In another embodiment of the invention, phosphatidylserine may beemployed as an alternative to phosphatidylethanolamine in preparationand use of therapeutic compounds, wherein the phospholipid is boundthrough the polar head group to a physiologically acceptable monomer orpolymer.

In another embodiment of the invention, phosphatidylcholine,phosphatidylinositol, phosphatidylglycerol, phosphatidic acidlysophospholipids, and related polar phospholipids may be employed as analternative to phosphatidylethanolamine in preparation and use oftherapeutic compounds, wherein the phospholipid is bound through thepolar head group to a physiologically acceptable monomer or polymer.When acylglycerols are used, such as monoacylglycerol, diacylglycerol,and triacylglycerol, the polar head group is a hydroxyl group. Otherlipids which enable the methods of the invention are sphingomyelin,sphingosine, and ceramide.

In another embodiment of the invention, glycerolipid derivatives bearingether or alkyl bonds instead of ester bonds at the C1 and C2 positionsof the glycerol backbone may be used as the therapeutic Lipid-conjugatecompound.

In another embodiment of the invention, the lipid-conjugates describedherein are used in a process for manufacture of a pharmaceuticalcomposition for treating a subject afflicted with a disorder of smoothmuscle cell proliferation, obstructive respiratory disease, lung injury,colitis, Crolin's disease, intestinal mucosal injury, central nervoussystem insult, ischemic/reperfusion injury, aterial stenosis andrestenosis, multiple sclerosis, sn diseases, contact dermatitis,seboreic dermatitis, psoriasis, conjunctivitis, cardiovascular disease,including prophylaxis for invasive procedures, atherosclerosis, invasivecellular proliferative disorders, primary cancer, metastatic cancer,hemolytic syndromes, sepsis, acute respiratory distress syndrome, tissuetransplant rejection syndromes, autoimmune disease, arthritis, viralinfection, HIV infection, chlamydia infection, or hypersensitivityconjunctivitis.

In another embodiment of the invention, the lipid-conjugates describedherein are used in a process for manufacture of a pharmaceuticalcomposition for the treatment of diseases which requires controllingphospholipase A2 activities, controlling the production aid/or action oflipid mediators, amelioration of damage to cell surface byglycosaminoglycans (GAG) and proteoglycans, controlling the productionof oxygen radicals and nitric oxide, protection of lipoproteins fromdamaging agents, anti-oxidant therapy; anti-endotoxin therapy;controlling of cytokine, chemokine and interleukine production,controlling the proliferation of cells, controlling of anigiogenesis andorgan vascularization; inhibition of invasion-promoting enzymes,controlling of cell invasion, controlling of white cell activation,adhesion and extravasation, amelioration of ischemia/reperfusion injury,inhibition of lymphocyte activation, controlling of blood vessel andairway contraction, protection of blood brain barrier, controlling ofneurotransmitter production and action or extracorporeal tissuepreservation.

In one embodiment, the invention provides a method of treating a subjectsuffering from an intestinal disease, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering froman intestinal disease.

In another embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject afflicted with an intestinal disease.

In another embodiment, the invention provides a pharmaceuticalcomposition for treating a subject suffering from an intestinal disease,including, inter alia, a lipid or phospholipid moiety bonded to aphysiologically acceptable monomer, dimer, oligomer, or polymer; and apharmaceutically acceptable carrier or excipient.

In one embodiment of the invention, the intestinal disease may be, interalia, Crohn's disease, ulcerative colitis, immuno-inflammatoryintestinal injury, drug-induced enteropathy, ischemia-induced intestinalinjury or any combination thereof.

BRIEF DESCRIPTION OF FIGURES

FIG. 1.1: Inhibition of endothelin-1 (ET)-induced contraction of rattracheal rings by Lipid-conjugates. A: Contraction of rat trachea byEndothelin-1. B: Effect of HyPE on ET-induced contraction of rattrachea.

FIG. 1.2: Effect of HyPE and Hyaluronic acid (HA) on ET-1 inducedcontraction of rat trachea.

FIG. 1.3: Effect of HyPE and Hyaluronic acid (HA) on Acetylcholine(AcCh)-induced contraction of isolated rat trachea rings.

FIG. 1.4: Lipid-conjugates ameliorate respiratory function in asthmaticrats.

FIG. 1.5: Amelioration of respiratory function in ovalbumin-challengedasthmatic rats by aerosolic administration of HyPE.

FIG. 1.6: Amelioration of airway remodeling in ovalbumin-sensitizedasthmatic rats by inhalation of HyPE (compared with systemicdexamethasone treatment).

FIG. 2.1: Amelioration of intestinal permeability in rats withindomethacin-induced small intestinal injury by CMPE.

FIG. 2.2: Amelioration of indomethacin-induced small intestinal damageby CMPE; macroscoring (left panel) and histological score (right panel).

FIG. 2.3: Amelioration of intestinal permeability in rats withTNBS-induced colitis by CMPE.

FIG. 2.4: CMPE suppresses phospholipase A₂ (PLA₂) activity in plasma ofrats with TNBS-induced colitis.

FIG. 2.5: Amelioration of TNBS-induced colon damage by treatment withCMPE: Histology micrographs.

FIG. 2.6: Amelioration of TNBS-induced colon damage by treatment withCMPE: Histological morphometry.

FIG. 2.7: HyPE (administered orally) ameliorates dextran sulfate-inducedcolitis in mice: Pathological score.

FIG. 2.8: HyPE (administered orally) abates colon shortening in micewith dextran sulfate-induced colitis.

FIG. 3.1: Lipid-conjugates inhibit the secretion of PGE₂ from glialcells stimulated by LPS.

FIG. 3.2: Lipid-conjugates inhibit the secretion of PGE₂ from glialcells stimulated by pardaxin (PX).

FIG. 3.3: Lipid-conjugates inhibit the production of nitric oxide byLPS-stimulated rat glial cells.

FIG. 3.4: Lipid-conjugates inhibit the production of nitric oxide byPX-stimulated PC12 cells.

FIG. 3.5: Lipid-conjugates inhibit the secretion of sPLA₂ from glialcells stimulated by LPS.

FIG. 3.6: Lipid-conjugates inhibit PX-induced activation of PLA2(expressed as fatty acid release) in PC12 cells.

FIG. 3.7: Effect of CMPE on LPS-induced OA release.

FIG. 3.8: Lipid-conjugates inhibit PX-induced dopamine release by PC12cells.

FIG. 3.9: Lipid-conjugates inhibit PX-induced production of 5-HETE byPC12 cells.

FIG. 3.10: Effect of Lipid-conjugates on T-cell permeation through amonolayer of endothelial cells.

FIG. 5.1: Effect of CMPE on the proliferation of cultured humanpsoriatic fibroblasts and Swiss 3T3 cells.

FIG. 6.1: Effect of Lipid-conjugates on LDL-endogenous phospholipase A₂activity.

FIG. 6.2: Effect of HyPE on uptake of oxidized LDL (ox LDL).

FIG. 7.1: Effect of HyPE on bovine aortic smooth muscle cell (SMC)proliferation.

FIG. 7.2: Effect of HyPE on proliferation of bovine aortic SMCs,stimulated with thrombin (48 hours).

FIG. 7.3: Effect of Lipid-conjugates on proliferation of human venoussmooth muscle cells.

FIG. 74: Effect of Lipid-conjugates on ischemia/reperfusion—inducedleukocyte adhesion (A) and extravasation (B) in rat cremaster muscle.

FIG. 7.5: Effect of Lipid-conjugates on red blood cell (RBC) adhesion toactivated endothelial cells (EC).

FIG. 8.1: Effect of Lipid-conjugates on secretion of collagenase IV(MMP-2) by human fibrosarcoma cells.

FIG. 8.2: HyPE inhibits hyaluronic acid degradation by hyaluronidase.

FIG. 8.3: Effect of Lipid-conjugates on the activity of exogenousheparinase.

FIG. 8.4: Effect of Lipid-conjugates on invasiveness of humanfibrosarcoma cells

FIG. 8.5: Effect of Lipid-conjugates on proliferation of bovine aorticendothelial cells (EC).

FIG. 8.6: Effect of HyPE on proliferation of human bone marrowendothelial cells (HBMEC) induced by growth factors.

FIG. 8.7: Effect of Lipid-conjugates on growth factor-induced capillaryformation by HNMEC in fibringel

FIG. 8.8: Effect of Lipid-conjugates on mouse lung metastases formationinduced by mouse melanoma cells.

FIG. 9.1: CMPE protects BGM cells from membrane lysis induced bycombined action of hydrogen peroxide (produced by glucose oxidase=GO),and exogenous phospholipase A₂ (PLA₂).

FIG. 9.2: CMP protects BGM cells from glycosaminoglycan degradation byHydrogen peroxide (produced by GO).

FIG. 9.3: HyPE protects LDL from copper-induced oxidation.

FIG. 11.1-I: Effect of lipid-conjugates on LPS-induced production ofTNFα in human whole blood.

FIG. 11.1-II: Effect of HyPE on LPS-induced production of TNFα in humanwhole blood.

FIG. 11.2: Effect of HyPE on rat survival in LPS-induced endotoxinemia.

FIG. 11.3: Effect of HyPE on serum levels of TNF-α and IL-6 in septicrats.

FIG. 11.4: Effect of HyPE on TNF-α production after i.p. administrationof LPS and simultaneous i.v. administration of HyPE.

FIG. 11.5: Effect of HyPE on serum cytokine levels in rats injected withLPS or LPS+LTA.

FIG. 11.6: Effect of HyPE on mRNA expression of IL-1, TNF-α and IL-6genes in lung and kidney of rats with LPS-induced sepsis.

FIG. 11.7: Effect of HyPE on mRNA expression of sPLA₂-IIA and iNOS genesin kidney and lung of rats with LPS-induced sepsis.

FIG. 11.8: Effect of HyPE on ICAM-1 expression in lung and kidney ofrats with LPS-induced sepsis.

FIG. 12.1: Effect of different Lipid-conjugates on LPS-induced IL-8production.

FIG. 12.2: Effect of HyPE on LPS-induced chemokine production.

FIG. 12.3: Effect of HyPE on LTA-induced IL-8 production.

FIG. 12.4: Effect of HyPE on LPS-induced ICAM-1 and E-selectinexpression.

FIG. 12.5: Effect of HyPE on LPS-induced activation of NF-kB in LMVEC.

FIG. 13.1: Inhibition of MHC-1 expression by IFN-γ stimulated humanumbilical vein endothelial cells (HUVEC) by HyPE.

FIG. 13.2: CMPE inhibits the proliferation of lymphocytes in vitro.

FIG. 13.3: Inhibition of MLR-induced proliferation of lymphocytes byHyPE.

FIG. 14-1: Effect of Lipid-conjugates on HIV infectivity.

FIG. 15.1: Effect of CMPE on allergic conjunctivitis in guinea pigs.Corneal opacities at the immediate post-provocation phase.

FIG. 15.2: Effect of CMPE on allergic conjunctivitis in guinea pigs.Corneal opacities at the late post-provocation phase

FIG. 15.3: Effect of CMPE on prostaglandin E₂ (PGE₂) and leukotriene B₄(LTB₄) levels in the cornea of guinea pigs with allergic conjunctivitis.

FIG. 16.1: Effect of Lipid-conjugates on injection of HeLa cells bychllamydia.

FIG. 16.2: Effect of Lipid-conjugates on chlamydia-induced apoptosis ofHeLa cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides lipid-conjugates which display a wide-rangecombination of cytoprotective pharmacological activities. Thesecompounds can alleviate airway obstruction in asthma, protect mucosaltissue in gastrointestinal disease, suppress immune responses, alleviatecutaneous hypersensitivity reactions, inhibit cell proliferationassociated with vascular injury and immunological responses, inhibitcell migration associated with vascular and central nervous systemdisease, attenuate oxidative damage to tissue proteins and cellmembranes, interfere with viral spread, reduce tissue destroying enzymeactivity, and reduce intracellular levels of chemokines and cytokines.Thus these compounds are useful in the treatment of a diversity ofdisease states, including obstructive respiratory disease, colitis,Crohn's disease, central nervous system insult, multiple sclerosis,contact dermatitis, psoriasis, cardiovascular disease, invasive medicalprocedures, invasive cellular proliferative disorders, anti-oxidanttherapy, hemolytic syndromes, sepsis, acute respiratory distresssyndrome, tissue transplant rejection syndromes, autoimmune disease,viral infection, and hypersensitivity conjunctivitis.

Obstructive respiratory disease is a disease of luminal passages in thelungs, marked by dyspnea, tachypnea, or ausculatory or radiologicalsigns of airway obstruction. While asthma is a prototypical disorder forobstructive respiratory disease, this condition is encounteredclinically also in acute pulmonary infections, acute respiratorydistress syndrome, and as chronic obstructive pulmonary disease. Thepathophysiology is attributed to obstruction of air flow due toconstriction of airway lumen smooth muscle and accumulation ofinfiltrates in and around the airway lumen.

Colitis is a chronic disease of the gastrointestinal lumen, marked byabdominal discomfort, diarrhea and, upon radiological or histologicaldiagnosis, characteristic signs of mucosal damage including epithelialdenudation. Crohn's disease is a related disorder affecting typicallythe small intestine but which may involve any region of thegastrointestinal tract.

Multiple sclerosis is a disease of white matter, marked by motorweakness or sensory disturbance, or both, usually diagnosed by spinalfluid analysis or magnetic resonance imaging. Visual disturbance,including blindness, is common as well. In regions of disease activity,the blood brain barrier is impaired.

Skin hypersensitivity reactions, otherwise known as contact dermatitis,are marked by external signs of tissue irritation such as localizedredness, swelling, and pruritis. Virtually any substance may produce thecondition, and it is one of the most common complaints diagnosed bydermatologists.

Psoriasis is also one of the most common dermatologic diseases,affecting 1 to 2 percent of people. The most common areas of involvementare the elbows, knees, gluteal cleft, and the scalp. In active lesionsof psoriasis, the rate of epidermal cell replications is accelerated.Long-term use of topical glucocorticoids is often accompanied by loss ofeffectiveness.

Cardiovascular disease refers to both disorders of blood vessel lumennarrowing as well as to resultant ischemic syndromes of the targetorgans they supply, such as heart, kidney, and brain. Ischemia, orreduced blood supply, results from the narrowing of a blood vessel. Thesigns and symptoms of cardiovascular disease include, among others,angina pectoris, weakness, dyspnea, transient ischemic attacks, stroke,and renal insufficiency. Diagnosis is based on clinical grounds inconjunction with ancilliary diagnostic tests, such as blood tests,electrocardiograms, echography, and angiography. Atherosclerosis is acommon element in cardiovasular disease in which narrowing of the bloodvessel lumen is due to scar-like plaques formed from reactive,migrating, and proliferating cells and from local incorporation of bloodfat, cholesterol, and lipoprotein. Of particular significance in thisrespect is the accumulation of low density lipoprotein (LDL), which maybe accelerated when damaged by oxidation. Plaques are considered to bethe sites for both acute and chronic stenotic lesions, wherein the riskof tissue ischemia rises.

Stenotic or narrowing lesions of blood vessels occur not only inatherosclerosis but in other systemic cardiovascular disorders as well.Among these are arterial hypertension, vasculitides, including thevasculitis associated with transplanted organs, and coagulativedisorders. Many of these disorders, particularly hypertension,atherosclerosis, and vasculitis occur concommitantly in the samepatient.

Reperfusion injury and ischemia/reperfusion injury refers to the tissueinjury and initiation of necrosis following the resumption of blood flowto a previously ischemic tissue. This phenomenon is recognized as animportant component of ischemic and post-ischemic types of injury,particularly to brain and heart tissue. One pathophysiological mechanismwhich predominates in reperfusion is the damaging effect of reactiveoxygen species, otherwise known as oxidative damage or free radialinjury. Nitric oxide and its radicals are also implicated in thepathophysiology. The production of these noxious chemical species isattributed to the local accumulation, adhesion, and transmigration ofleukocytes at the lesion site.

Invasive medical procedures, such as catheterization of arteries orveins or open surgery are frequently associated with tissue ischemia dueto blood vessel injury as well as to reperfusion injury, both of whichmay arise in the course of an invasive procedure. Thus preservation ofblood vessel potency and prevention of reperfusion injury are thesubject of intense investigation in medical science. Such procedures areperformed for both diagnostic and therapeutic purposes, and adjuvantdrugs are commonly prescribed to prevent complications of blood vesselinjury or restenosis. Formation of these lesions involves a multiplicityof participants, including coagulative elements of the blood, bloodcells, and the structural elements and cells of the blood vessel lumenwall. For example, arterial restenosis appearing after successfulballoon angioplasty is frequently due to the narrowing of the innerdiameter of the artery by the growth (proliferation) of smooth musclecells in the areas of irritation caused by the balloon angioplasty. Thisnew stenotic lesion may be comprised from other cell types as well,including leukocytes, accumulating at the lesion site through processesof migration and local proliferation. The two events (cell migration andproliferation) are almost certainly due to the coordinated interactionof a number of different cytokines likely released by early accumulationof macrophages at the site of original tissue injury. Thus leukocytescontribute to stenotic lesion formation through the processes ofmigration, local proliferation, passage through endothelial barriers,accumulation of cholesterol-rich lipoprotein, conversion to foam cells,and secretion of cytokines. This proliferation of cells and narrowing ofthe vascular lumen is not however restricted or limited to the coronaryarteries or cerebral circulation. It can also occur post-operativelycausing restenosis in, for example, peripheral vascular systems.

In the context of the present invention, the term cardiovascular diseaserefers to blood vessel lumen narrowing arising in the course ofatherosclerosis, vasculitis, invasive procedures, particularlycatheterization of an artery or vein, and the ischemic syndromesassociated with them.

Transplantation of tissue, grafts, and organs is frequently complicatedby the appearance of host-versus-graft and graft-versus-host disease,both of which may occur acutely or chronically in the recipient of thegraft. The source of the graft may be allogeneic (from the same species)or xenogeneic (from another species). Whether as complication due to theinduced hyperactive immune response, or through another mechanism,vasculitis remains a frequently encountered complication of tissuetransplantation procedures. Moreover, vascular damage due toreperifusion injury is considered to be a major factor in thepost-surgical malfunctioning of tissue and organ transplants.

Autoimmune diseases are conditions in which the change in clinical stateof the subject is attributed to aberrant cellular and/or humoral immuneresponses. The most common autoimmune diseases in the U.S. are juvenilediabetes, Hashimoto's and Grave's thryroiditis, rheumatoid arthritis,Crohn's disease and ulcerative colitis, chronic active hepatitis,vitaligo, glomerulonepluitis, uveitis, multiple sclerosis, scleroderma,hemolytic anemia, idiopathic thronibocytopenic purpura, myastheniagravis, systemic lupus erythematosis, and pemphigus.

Hyper-proliferative cellular disorders, such as cancer cells arising atprimary organ sites or at other loci of spread (metastases), are one ofthe leading causes of death in the U.S. Cancers are frequently highlyresistant to all forms of treatment including therapy with potentanti-proliferative drugs and radiation. Increasingly the medicalcommunity is becoming aware of the critical role played by thevasculature associated with both the primary and metastatic forms ofdisease. Like any cell cluster, cancer cells are dependent upon areliable blood supply and in fact, cancer cells are known to encouragethe process of de novo vascularization through elaboration of growthfactors which act on endothelial cells and smooth muscle cells to formnew blood vessels, thus supplying the cancerous growth.

Metastasis, the spread of cancer cells to ectopic sites, is frequently avasculature dependent process as well, often referred to as hematogenousspread. The physiological barrier imposed by the blood vessel wall,comprised from elements such as endothelial cells and basement membranesubstance, is normally highly selective to the passage of cells.However, metastatic cells abrogate this barrier, employing a variety ofmechanisms, some of which have been established in the scientificliterature. For example, such abnormal cells produce hydrolytic enzymeswhich degrade the extracellular matrix and associated components of thevascular barrier, such as collagenase, heparinase, and hyaluronidase.Thus a critical factor in the metastatic process is the ability ofcancer cells to intrude through or permeate the wall of the blood vessellumen, thus arriving to invade a new tissue site after travel throughthe circulation. Cancer cells also elaborate messenger chemicals, knownas cytokines and chemokines, which enable the metastatic process, frommany aspects, including angiogenesis.

Cellular elaboration of cytokines and chemokines serve an importantregulatory function in health; however, when a hyperactive response tostress or disease is triggered, these compounds may present in excessand damage tissue, thereby pushing the disease state toward furtherdeterioration. Cytokine overproduction is involved in numerous diseases,such as sepsis, airway and lung injury, renal failure, transplantrejection, skin injuries, intestine injuries, cancer development andmetastasis, central nervous sytem disorders, vaginal bacterialinfection, and more. Two examples in which this occurs are systemicinfection, in particular when due to blood born bacteria (septicemia),and in the pulmonary condition known as acute (or adult) respiratorydistress syndrome (ARDS). In ARDS, lung spaces fill with fluid, impedinggas exchange and producing respiratory failure. Although plateletaggregation occurs, the major offenders appear to be monocyticphagocytes and leukocytes that adhere to endothelial surfaces andundergo a respiratory burst to inflict oxidant injury and releasechemokines such as Gro α, ENA-78, CX3X and MCP-1, in addition toleukotrienes, thromboxanes, and prostaglandins. The monocyticphagocytes, mainly macrophages in the alveoli and those lining thevasculature, also release oxidants, mediators, and a series ofdegradative enzymes that directly damage endothelial cells and causeleukocytes to release their lysosomal enzymes. The mortality rate isover 50%. The most common causes of ARDS are infection, aspiration,smoke and toxin inhalation, as well as systemic processes initiatedoutside the lung, including bacterial septicemia. The sepsis syndromeand shock are triggered by the interactions of various microbialproducts in the blood, in particular, gram-negative endotoxins, withhost mediator systems. The incidence is estimated to be up to 500,000cases per year in the U.S. alone, a Fig. which is considered to rise dueto the increasing prevalence of antibiotic resistant organisms. Avariety of host mediators have been implicated in the pathogenesis ofsepticemia and septic shock (referred to collectively herein as sepsis)including factors released from stimulated cells, in particular,cytokines, tumor necrosis factor-α (TNF), Gro α, ENA-78, CX3X and MCP-1,NFκβ transcription factor, lysosomal enzymes and oxidants fromleukocytes, and products of the metabolism of arachidonic acid, amongothers.

Red blood cell lysis, or hemolysis, may be an inherited or acquireddisorder, giving rise to anemia, iron deficiency, or jaundice. Among theacquired syndromes are membrane anomalies due to direct toxic effects ofsnake bites or of infectious agents, including viral, bacterial andparasitic etiologies, particularly malaria; exposure to oxidizingsubstances through ingestion or disease; or as a result of mechanicaltrauma within abnormal blood vessels. This latter condition, known asmicroangiopathic hemolysis, is considered to be related in mechanism tothe hemolysis produced from blood passage through prosthetic implants,such as heart valves. Inherited red blood cell membrane fragility oftenoccurs due to intracorpuscular enzyme and structural defects, such asglucose 6-phosphatase deficiency, sickle cell anemia, and thalessemia.Red blood cell lysis is one of the limiting factors in the storage lifeof blood products, particularly when subjected to free-radical formingphotodynamic virocidal treatments, such as γ-irradiation.

The acquired immunodeficiency syndrome is considered to be a rapidlygrowing global epidemic and one route of spread is through contaminatedblood products. Transmission and progression of this disease isdependent upon the infective activity of the human immunodeficiencyvirus. Current therapies are limited primarily to the administration ofreverse transcriptase inhibitors, drugs of high expense and low patienttolerability.

Oxidative injury refers to the effect of peroxidation and free radicalproduction on body tissues. To some extent, peroxide production is anormal, physiological process, attributed, for example, a role in immunedefense. However, in stress and disease states, or over the naturalcourse of time, as in physiological senesence, the accumulative additionof these unstable chemical moieties to tissue structures, includingmembrane components and blood proteins, leads to an irreversible patternof injury. Agents that act as anti-oxidants can protect againstoxidative damage. Such protection has been the subject of numerousscientific publications.

Intracellular bacterial parasites are one of the most prevelant forms ofsexually transmitted disease and are frequently intractable toconventional antibiotic therapy. Vaginal infection with chlamydiaspecies is a salient example.

In one embodiment, the present invention offers methods for thetreatment of disease based upon administration of lipids covalentlyconjugated through their polar head group to a physiologicallyacceptable chemical moiety, which may be of high or low molecularweight.

In one embodiment, the lipid compounds (Lipid-conjugates) of the presentinvention are described by the general formula:[phosphatidylethanolamine-Y]n-X[phosphatidylserine-Y]n-X[phosphatidylcholine-Y]n-X[phosphatidyl inositol-Y]n-X[phosphatidylglycerol-Y]n-X[phosphatidic acid-Y]n-X[lyso-phospholipid-Y]n-X[diacyl-glycerol-Y]n-X[monoacyl-glycerol-Y]n-X[sphingomyelin-Y]n-X[sphingosine-Y]n-X[ceramide-Y]n-Xwherein

-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms; and-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer; and-   n, the number of lipid molecules bound to X, is a number from 1 to    1000.

In one embodiment of this invention, n is a number from 1 to 1000. Inanother embodiment, n is a number from 1 to 500. In another embodiment,n is a number from 1 to 100. In another embodiment, n is a number from100 to 300. In another embodiment, n is a number from 300 to 500. Inanother embodiment, n is a number from 500 to 800.

In one embodiment, the lipid compounds of this invention, known hereinas lipid conjugates (Lipid-conjugates) are now disclosed to possess acombination of multiple and potent pharmacological effects in additionto the ability to inhibit the extracellular form of the enzymephospholipase A2. The set of compounds comprisingphosphatidylethanolamine covalently bound to a physiologicallyacceptable monomer or polymer, is referred to herein as thePE-conjugates. Related derivatives, in which either phosphatidylserine,phosphatidylcholine, phosphatidylinositol, phosphatidic acid orphosphatidylglycerol are employed in lieu of phosphatidylethanolamine asthe lipid moiety provide equivalent therapeutic results, based upon thebiological experiments described below for the Lipid-conjugates and thestructural similarities shared by these compounds. Other Lipid-conjugatederivatives relevant to this invention are Lipid-conjugates wherein atleast one of the fatty acid groups of the lipid moieties at position C1or C2 of the glycerol backbone are substituted by a long chain alkylgroup attached in either ether or alkyl bonds, rather than esterlinkage.

As defined by the structural formulae provided herein for theLipid-conjugates, these compounds may contain between one to onethousand lipid moieties bound to a single physiologically acceptablepolymer molecule.

Administration of the Lipid-conjugates in a diversity of animal and cellmodels of disease invokes remarkable, and unexpected, cytoprotectiveeffects, which are useful in the treatment of disease. They are able tostabilize biological membranes; inhibit cell proliferation; suppressfree radical production; suppress nitric oxide production; reduce cellmigration across biological barriers; influence chemokine levels,including MCP-1, ENA-78, Gro α, and CX3C; affect gene transcription andmodify the expression of MHC antigens; bind directly to cell membranesand change the water structure at the cell surface; inhibit the uptakeof oxidized lipoprotein; prevent airway smooth muscle constriction;suppress neurotransmitter release; reduce expression of tumor necrosisfactor-α (TNF-α); modify expression of transcription factors such asNFκB; inhibit extracellular degradative enzymes, including collagenase,heparinase, hyaluronidase, in addition to that of PLA2; and inhibitviral infection of white cells. Thus the Lipid-conjugates providefar-reaching cytoprotective effects to an organism suffering from adisease wherein one or more of the presiding pathophysiologicalmechanisms of tissue damage entails either oxidation insult giving riseto membrane fragility; hyperproliferation behavior of cells giving riseto stenotic plaque formation in vascular tissue, angiogenesis and benignor malignant cancer disease, or psoriasis; aberrant cell migrationgiving rise to brain injury or tumor cell metastases; excessiveexpression of chemokines and cytokines associated with central nervoussystem (CNS) insult, sepsis, ARDS, or immunological disease; cellmembrane damage giving rise to CNS insult, CVS disease, or hemolysis;peroxidation of blood proteins and cell membranes giving rise toatherosclerosis or reperfusion injury; excessive nitric oxide productiongiving rise to CNS insult, reperfusion injury, and septic shock;interaction with major histocompatability antigens (MHC) associated withautoimmune diseases and alloimmune syndromes, such as transplantrejection.

In one embodiment of the present invention, the useful pharmacologicalproperties of the lipid or Lipid-conjugates may be applied for clinicaluse, and disclosed herein as methods for treatment of a disease. Thebiological basis of these methods may be readily demonstrated bystandard cellular and animal models of disease as described below.

While pharmacological activity of the Lipid-conjugates described hereinmay be due in part to the nature of the lipid moiety, the multiple anddiverse combination of pharmacological properties observed for theLipid-conjugates emerges ability of the compound structure to actessentially as several different drugs in one chemical entity. Thus, forexample, internal mucosal injury, as may occur in colitis or Crohn'sdisease, may be attenuated by any one or all of the pharmaceuticalactivities of immune suppression, anti-inflammation, anti-oxidation,nitric oxide production, or membrane stabilization. Protection of bloodvessels from periluminal damage, as may occur in atherosclerosis, mayentail activity from anti-proliferative, anti-chemokine, antioxidant, orantimigratory effects. Treatment of obstructive respiratory disease mayinvolve any one of the many activities of the Lipid-conjugates rangingfrom suppression of nitric oxide, anti-chemokine, anti-proliferative, ormembrane stabilization effects.

Proliferation of vascular tissue is an element of both the atherogenesisof sclerotic plaques as well as a feature of primary and metastaticcancer lesion growth. Stabilization of biological membranes may preventhemolysis as well as mucosal bowel injury. Attenuation of chemokinelevels may ameliorate ARDS as well as militate against atherogenesis.Anti-oxidant activity protects may protect against reperfusion injuryand ischemia/reperfusion injury as well as CNS insult, atherosclerosis,and hemolysis. These and other advantages of the present invention willbe apparent to those skilled in the art based on the followingdescription.

The use of a single chemical entity with potent anti-oxidant,membrane-stabilizing, anti-proliferative, anti-chemokine,anti-migratory, and anti-inflammatory activity provides increasedcytoprotection relative to the use of several different agents each witha singular activity. The use of a single agent having multipleactivities over a combination or plurality of different agents providesuniform delivery of an active molecule, thereby simplifying issues ofdrug metabolism, toxicity and delivery. The compounds of the presentinvention also exhibit properties present only in the combined molecule,not in the individual components.

In one embodiment, the compounds of the invention may be used for acutetreatment of temporary conditions, or may be administered chronically,especially in the case of progressive, recurrent, or degenerativedisease. In one embodiment of the invention, the concentrations of thecompounds will depend on various factors, including the nature of thecondition to be treated, the condition of the patient, the route ofadministration and the individual tolerability of the compositions.

In another embodiment, the invention provides low-molecular weightLipid-conjugates, previously undisclosed and unknown to possesspharmacological activity, of the general formula:[Phosphatidylethanolamine-Y]n-X[Phosphatidylserine-Y]n-X[Phosphatidylcholine-Y]n-X[Phosphatidylinositol-Y]n-X[Phosphatidylglycerol-Y]n-X[Phosphatidic acid-Y]n-X[lyso-phospholipid-Y]n-X[diacyl-glycerol-Y]n-X[monoacyl-glycerol-Y]n-X[sphingomyelin-Y]n-X[sphingosine-Y]n-X[ceramide-Y]n-Xwherein

-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms; and-   X is salicylate, salicylic acid, aspirin, a monosaccharide,    lactobionic acid, maltose, an amino acid, glycine, carboxylic acid,    acetic acid, butyric acid, dicarboxylic acid, glutaric acid,    succinic acid, fatty acid, dodecanoic acid, didodecanoic acid, bile    acid, cholic acid, cholesterylhemmisuccinate, a dipeptide, a    disaccharide, a trisaccharide, an oligosaccharide, an oligopeptide,    or a di- or trisaccharide monomer unit of heparin, heparan sulfate,    keratin, keratan sulfate, chondroitin, chondoitin-6-sulfate,    chondroitin-4-sulfate, dermatin, dermatan sulfate, dextran, or    hyaluronic acid, a glycosaminoglycan, polygeline (‘haemaccel’),    alginate, hydroxyethyl starch (hetastarch), polyethylene glycol,    polycarboxylated polyethylene glycol, chondroitin-6-sulfate,    chondroitin-4-sulfate, keratin, keratin sulfate, heparan sulfate,    dermatin, dermatan sulfate, carboxymethylcellulose, heparin,    dextran, or hyaluronic acid; and n, the number of lipid molecules    bound to X, is a number from 1 to 1000.

In one embodiment of this invention, n is a number from 1 to 1000. Inanother embodiment, n is a number from 1 to 500. In another embodiment,n is a number from 1 to 100. In another embodiment, n is a number from100 to 300. In another embodiment, n is a number from 300 to 500. Inanother embodiment, n is a number from 500 to 800.

In another embodiment of the invention, these Lipid-conjugatederivatives possess wide-spectrum pharmacological activity and, aspharmaceutical agents administered to treat disease, are consideredanalogous to the Lipid-conjugates comprised from high molecular weightpolymers. Other lipid-conjugate derivatives relevant to this inventionare glycerolipid moieties in which at least one of the two long chainalkyl groups in position C1 and C2 of the glycerol backbone are attachedin ether or alkyl bonds, rather than ester linkage.

The present invention is further illustrated in the following examplesof the therapeutic Lipid-conjugate compounds, their chemicalpreparation, their anti-disease activity, and methods of use aspharmaceutical compositions in the treatment of disease.

Preferred Compounds

In the methods, according to embodiments of the invention, theLipid-conjugates administered to the subject are comprised from at leastone lipid moiety covalently bound through an atom of the polar headgroup to a monomer or polymeric moiety (referred to herein as theconjugated moiety) of either low or high molecular weight. When desired,an optional bridging moiety can be used to link the Lipid-conjugatesmoiety to the monomer or polymeric moiety. The conjugated moiety may bea low molecular weight carboxylic acid, dicarboxylic acid, fatty acid,dicarboxylic fatty acid, acetyl salicylic acid, cholic acid,cholesterylhemisuccinate, or mono- or di-saccharide, an amino acid ordipeptide, an oligopeptide, a glycoprotein mixture, a di- ortrisaccharide monomer unit of a glycosaminoglycan such as a repeatingunit of heparin, heparan sulfate, hyaluronic acid, chondrotin-sulfate,demmatan, keratan sulfate, or a higher molecular weight peptide oroligopeptide, a polysaccharide, polyglycan, protein, glycosaminoglycan,or a glycoprotein mixture. From a composition aspect,phospholipid-conjugates of high molecular weight, and associatedanalogues, are the subject of U.S. Pat. No. 5,064,817, as well as thepublications cited herein.

In one embodiment of the invention, when the conjugated carrier moietyis a polymer, the ratio of lipid moieties covalently bound may rangefrom one to one thousand lipid residues per polymer molecule, dependingupon the nature of the polymer and the reaction conditions employed. Forexample, the relative quantities of the starting materials, or theextent of the reaction time, may be modified in order to obtainLipid-conjugate products with either high or low ratios of lipidresidues per polymer, as desired.

The term “moiety” means a chemical entity otherwise corresponding to achemical compound, which has a valence satisfied by a covalent bond.

Examples of polymers which can be employed as the conjugated moiety forproducing Lipid-conjugates for use in the methods of this invention maybe physiologically acceptable polymers, including water-dispersible or-soluble polymers of various molecular weights and diverse chemicaltypes, mainly natural and synthestic polymers, such asglycosaminoglycans, hyaluronic acid, heparin, heparin sulfate,chondrotin sulfate, chondrotin-6-sulfate, chondroitin-4-sulfate, keratinsulfate, dermatin, sulfate, plasma expanders, including polygeline(“Haemaccel”, degraded gelatin polypeptide crosslinked via urea bridges,produced by “Behring”), “hydroxyethylstarch” (Htastarch, HES) andextrans, food and drug additives, soluble cellulose derivatives (e.g.,methylcellulose, carboxymethylcellulose), polyaminoacids, hydrocarbonpolymers (e.g., polyethylene), polystyrenes, polyesters, polyamides,polyethylene oxides (e.g. polyethyleneglycols,polycarboxyethyleneglycol), polyvinnylpyrrolidones, polysaccharides,alginates, assimilable gums (e.g., xanthan gum), peptides, injectableblood proteins (e.g., serum albumin), cyclodextrin, and derivativesthereof.

Examples of monomers, dimers, and oligomers which can be employed as theconjugated moiety for producing Lipid-conjugates for use in the methodsof the invention may be mono- or disaccharides, carboxylic acid,dicarboxylic acid, fatty acid, dicarboxylic fatty acid, acetyl salicylicacid, cholic acid, cholesterylhemisuccinate, and di- and trisaccharideunit monomers of glycosaminoglycans including heparin, heparan sulfate,hyaluronic acid, chondrotin, chondroitin-6-sulfate,chondroitin-4-sulfate, dermatin, dermatan sulfate, keratin, keratansulfate, or dextran.

In some cases, according to embodiments of the invention, the monomer orpolymer chosen for preparation of the Lipid-conjugate may in itself haveselect biological properties. For example, both heparin and hyaluronicacid are materials with known physiological functions. In the presentinvention, however, the Lipid-conjugates formed from these substances asstarting materials display a new and wider set of pharmaceuticalactivities than would be predicted from administration of either heparinor hyaluronic acid which have not been bound by covalent linkage to aphospholipid. It can be shown, by standard comparative experiments asdescribed below, that phosphatidylethanolamine (PE) linked tocarboxymethylcellulose (referred to as CMPE, CMC-Peor CME), tohyaluronic acid (referred to as HYPE, HyPE, and Hyal-PE), to heparin(referred to as HEPPE, HepPE, HePPE, Hepa-PE), to chondroitine sulfate A(referred to as CSAPE, CsaPE, CsAPE), to Polygeline (haemaccel)(referred to HemPE, HEMPE), or to hydroxyethylstarch (referred to asHesPE, HESPE), are far superior in terms of potency and range of usefulpharmaceutical activity to the free conjugates (the polymers above andthe like). In fact, these latter substances are, in general, notconsidered useful in methods for treatment of most of the diseasesdescribed herein, and for those particular cases wherein their use ismedically prescribed, such as ischemic vascular disease, theconcentrations for their use as drugs are are several orders ofmagnitude higher. Thus, the combination of a phospholipid such asphosphatidylethanolamine, or related phospholipids which differ withregard to the polar head group, such as phosphatidylserine (PS),phosphatidylcholine (PC), phosphatidylinositol (PI), andphosphatidylglycerol (PG), results in the formation of a compound whichhas novel pharmacological properties when compared to the startingmaterials alone.

The biologically active lipid conjugates described herein can have awide range of molecular weight, e.g., above 50,000 (up to a few hundredthousands) when it is desirable to retain the Lipid conjugate in thevascular system and below 50,000 when targeting to extravascular systemsis desirable. The sole limitation on the molecular weight and thechemical structure of the conjugated moiety is that it does not resultin a Lipid-conjugate devoid of the desired biological activity, or leadto chemical or physiological instability to the extent that theLipid-conjugate is rendered useless as a drug in the method of usedescribed herein.

In one embodiment, the invention provides a compound represented by thestructure of the general formula (A):

wherein

-   L is a lipid or a phospholipid;-   Z is either nothing, ethanolainine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein X is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between L, Z, Y and X is either an amide or an    esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (I):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms; and    -   X is either a physiologically acceptable monomer, dimer,        oligomer or a physiologically acceptable polymer, wherein X is a        glycosaminoglycan; and    -   n is a number from 1 to 1,000;    -   wherein if Y is nothing the phosphatidylethanolamine is directly        linked to X via an amide bond and if Y is a spacer, the spacer        is directly linked to X via an amide or an esteric bond and to        the phosphatidylethanolamine via an amide bond.

Preferred compounds for use in the methods of the invention comprise oneof the following as the conjugated moiety X: acetate, butyrate,glutarate, succinate, dodecanoate, didodecanoate, maltose, lactobionicacid, dextran, alginate, aspirin, cholate, cholesterylhemisuccinate,carboxymethyl-cellulose, heparin, hyaluronic acid, polygeline(haemaccel), polyethyleneglycol, and polycarboxylated polyethyleneglycol. The polymers used as starting material to prepare thePE-conjugates may vary in molecular weight from 1 to 2,000 kDa.

Examples of phosphatidylethanolamine (PE) moieties are analogues of thephospholipid in which the chain length of the two fatty acid groupsattached to the glycerol backbone of the phospholipid varies from 2-30carbon atoms length, and in which these fatty acids chains containsaturated and/or unsaturated carbon atoms. In lieu of fatty acid chains,alkyl chains attached directly or via an ether linkage to the glycerolbackbone of the phospholipid are included as analogues of PE. Accordingto the present invention, a most preferred PE moiety isdipalmitoylphosphatidy-ethanolamine.

Phosphatidyl-ethanolamine and its analogues may be from various sources,including natural, synthetic, and semisynthetic derivatives and theirisomers.

Phospholipids which can be employed in lieu of the PE moiety areN-methyl-PE derivatives and their analogues, linked through the aminogroup of the N-methyl-PE by a covalent bond; N,N-dimethyl-PE derivativesand their analogues linked through the amino group of theN,N-dimethyl-PE by a covalent bond, phosphatidylserine (PS) and itsanalogues, such as palmitoyl-stearoyl-PS, natural PS from varioussources, semisynthetic PSs, synthetic, natural and artifactual PSs andtheir isomers. Other phospholipids useful as conjugated moieties in thisinvention are phosphatidylcholine (PC), phosphatidylinositol (PI),phosphatidic acid and phosphoatidylglycerol (PG), as well as derivativesthereof comprising either phospholipids, lysophospholipids,phosphatidyic acid, sphingomyelins, lysosphingomyelins, ceramide, andsphingosine.

For PE-conjugates and PS-conjugates, the phospholipid is linked to theconjugated monomer or polymer moiety through the nitrogen atom of thephospholipid polar head group, either directly or via a spacer group.For PC, PI, and PG conjugates, the phospholipid is linked to theconjugated monomer or polymer moiety through either the nitrogen or oneof the oxygen atoms of the polar head group, either directly or via aspacer group.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (II):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein if Y is nothing the phosphatidylserine is directly linked to    X via an amide bond and if Y is a spacer, the spacer is directly    linked to X via an amide or an esteric bond and to the    phosphatidylserine via an amide bond.

In another embodiment, the compound according to the invention be[phosphatidylserine-Y]n-X, wherein Y is either nothing or a spacer groupranging in length from 2 to 30 atoms, X is a physiologically acceptablemonomer, dimer, oligomer or polymer wherein x is a glycosaminoglycan,and n is a number from 1 to 1000, wherein the phosphatidylserine may bebonded to Y or to X, if Y is nothing, via the COO⁻ moiety of thephosphatidylserine.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (III):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phosphatidyl, Z, Y and X is either an    amide or anesteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (IV):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (V):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (VI):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsatuiated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (VII):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond.

In one embodiment of the invention, phosphatidylcholine (PC),Phosphatidylinositol (PI), phosphatidic acid (PA), wherein Z is nothing,and Phosphatidylglycerol (PG) conjugates are herein defined as compoundsof the general formula (III).

In one embodiment of the invention Y is nothing. Non limiting examplesof suitable divalent groups forming the optional bridging group (spacer)Y, according to embodiments of the invention, are straight or branchedchain alkylene, e.g., of 2 or more, preferably 4 to 30 carbon atoms,—CO-alkylene-CO, —NH-alkylene-NH—, —CO-alkylene-NH—,—NH-alkylene-NHCO-alkylene-NH—, an amino acid, cycloalkylene, whereinalkylene in each instance, is straight or branched chain and contains 2or more, preferably 2 to 30 atoms in the chain, —(—O—CH(CH₃)CH₂—)_(x)—wherein x is an integer of 1 or more.

According to embodiments of the invention, in addition to thetraditional phospholipid structure, related derivatives for use in thisinvention are phospholipids modified at the C1 or C2 position to containan ether or alkyl bond instead of an ester bond. In one embodiment ofthe invention, the alkyl phospholipid derivatives and ether phospholipidderivatives are exemplified herein.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (VIII):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (IX):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (IXa):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (IXb):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (X):

-   -   wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;

-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;

-   Z is either nothing, ethanolaiine, serine, inositol, choline, or    glycerol;

-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;

-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and

-   n is a number from 1 to 1000;    wherein any bond between the ceramide phosphoryl, Z, Y and X is    either an amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XI):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is nothing;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein if Y is nothing the sphingosyl is directly linked to X via    an amide bond and if Y is a spacer, the spacer is directly linked to    X and to the sphingosyl via an amide bond and to X via an amide or    an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XII):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   L is ceramide;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the ceramide, Z, Y and X is either an amide    or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XIII):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the diglyceryl, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XIV):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the glycerolipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XV):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the glycerolipid, Z, Y and X is either an    amide or an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XVI):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XVII):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XVIII):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XIX):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XX):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond.

In another embodiment, the compound according to the invention isrepresented by the structure of the general formula (XXI):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond.

In one embodiment of the invention, the glycosaminoglycan may be, interalia, hyaluronic acid, heparin, heparan sulfate, chondrotin sulfate,keratin, keratan sulfate, dermatan sulfate or a derivative thereof.

In another embodiment, the glycosaminoglycan is di- and trisaccharideunit monomers of glycosaminoglycans. In another embodiment, thechondroitin sulfate may be, inter alia, chondroitin-6-sulfate,chondroitin-4-sulfate or a derivative thereof.

In one embodiment of the invention, the sugar rings of theglycosaminoglycan are intact. In another embodiment, intact refers toclosed. In another embodiment, intact refers to natural. In anotherembodiment, intact refers to unbroken.

In one embodiment of the invention, the structure of the lipid orphospholipids in any compound according to the invention is intact. Inanother embodiment, the natural structure of the lipid or phospholipidsin any compound according to the invention is maintained.

In one embodiment, the compounds according to the invention arebiodegradable.

In one embodiment, the compound according to the invention is a ompoundrepresented by the structure of the general formula (A):

wherein

-   L is phosphatidyl;-   Z is ethanolamine, wherein L and Z are chemically bonded resulting    in phosphatidylethanolamine;-   Y is nothing;-   X is hyaluronic acid; and-   n is a number from 1 to 1000;    wherein any bond between the phosphatidylethanolamine and the    hyaluronic acid is an amide bond.

In one embodiment, the compound according to the invention is a ompoundrepresented by the structure of the general formula (A):

wherein

-   L is phosphatidyl;-   Z is ethanolamine, wherein L and Z are chemically bonded resulting    in phosphatidylethanolamine;-   Y is nothing;-   X is chondroitin sulfate; and-   n is a number from 1 to 1000;    wherein any bond between the phosphatidylethanolamine and the    chondroitin sulfate is an amide bond.

Illustrative of preferred Lipid-conjugates for use in the methodsaccording to embodiments of this invention are those in which thelipid/phospholipid moiety is linked directly or indirectly through abridging moiety listed below. phospholipid spacer polymer (m.w.)abbreviation PE Dicarboxylic Polygeline (haemaccel) HeMPE; HemPE acid +(4-40 kDa) Diamine PE None Carboxymethylcellulose CMPE; CMC- (20-500kDa) PE PE None Hyaluronic acid HYPE (HyPE) (2-2000 kDa) PE DipalmitoicHyaluronic acid HYPE- acid (2-2000 kDa) dipalmitoyl PE None Polyethyleneglycol PE Y Hydroxyethylstarch HESPE; HesPE PE Dicarboxylic DextranDexPE acid + (1-2,000 kDa) Diamine PE None Dextran DexPE (1-2,000 kDa)PE None Albumin PE None Alginate (2-2000 kDa) PE None Polyaminoacid PENone Lactobionic acid PE None Acetylsalicylate PE None Cholesteryl-hemmisuccinate PE None Maltose PE Y None Cholic acid PE NonePolycarboxylated polyethylene glycol PE None Heparin HEPPE; HEPE;(0.5-110 kDa) HepPE Dimyristoyl- Y Variable DMPE PE Dimyristoyl- YHyaluronic acid HyDMPE PE PS Y Polygeline (haemaccel) PS Y Heparin PS YHyaluronic acid PC Y Polygeline (haemaccel) PC Y Heparin PC Y Hyaluronicacid PI Y Polygeline (haemaccel) PI Y Heparin PI Y Hyaluronic acid PG YPolygeline (haemaccel) PG Y Heparin PE Y Chondoitin sulfates CSPE PE YPolygeline (haemaccel) PG Y Hyaluronic acid

In one embodiment of the invention, the compounds administered are HyPE,CSAPE, CMPE, HemPE, HesPE, DexPE and As-PE. and pharmaceuticallyacceptable salts thereof, in combination with a physiologicallyacceptable carrier or solvent These polymers, when chosen as theconjugated moiety, may vary in molecular weights from 200 to 2,000,000Daltons. Various molecular weight species have been shown to have thedesired biological efficacy, as shown in the section below.

In addition to the compounds of the Examples, further illustrativecompounds of this invention are set forth in the section below.

Novel Compounds

Low molecular weight Lipid-conjugates, in which the conjugated moiety isa monomer such as a salicylate, a bile acid, orcholesterylheinmisuccinate, or a di- or trisaccaharide unit monomer of apolyglycosoaminoglycan such as heparin, heparan sulfate,chondrotin-6-sulfate, chondroitin-4-sulfate, hyaluronic acid, keratin,keratan sulfate, demiatin, or dermatan sulfate, have not been describedbefore. According to embodiments of the invention, these new compoundsdisplay a similar biological activity profile as demonstrated below forthe other Lipid-conjugates and have the general formula[Phosphatidylethanolamine-Y]n-X[Phosphatidylserine-Y]n-X[Phosphatidylcholine-Y]n-X[Phosphatidylinositol-Y]n-X[Phosphatidylglycerol-Y]n-X[Phosphatidic acid-Y]n-X[lyso-phospholipid-Y]n-X[diacyl-glycerol-Y]n-X[monoacyl-glycerol-Y]n-X[sphingomyelin-Y]n-X[sphingosine-Y]n-X[ceramide-Y]n-Xwherein

-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a mono- or disaccharide, carboxylated disaccharide, mono- or    dicarboxylic acids, a salicylate, salicylic acid, aspirin,    lactobionic acid, maltose, an amino acid, glycine, acetic acid,    butyric acid, dicarboxylic acid, glutaric acid, succinic acid, fatty    acid, dodecanoic acid, didodecanoic acid, bile acid, cholic acid,    cholesterylhemmisuccinate, a di- or tripeptide, an oligopeptide, a    trisacharide, or a di- or trisaccharide monomer unit of lieparin,    heparan sulfate, keratin, keratan sulfate, chondroitin,    chondoitin-6-sulfate, chondroitin-4-sulfate, dermatin, dermatan    sulfate, dextran, or hyaluronic acid; and-   n is the number of lipid moiety molecules bound to a molecule of X    wherein n is a number from 1 to 1000.

In one embodiment of this invention, low molecular weightphosphatidylethanolamine (PE)-conjugates are defined hereinabove as thecompounds of formula (1) wherein:

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a mono- or disaccharide, carboxylated disaccharide, mono- or    dicarboxylic acids, a salicylate, salicylic acid, aspirin,    lactobionic acid, maltose, an amino acid, glycine, acetic acid,    butyric acid, dicarboxylic acid, glutaric acid, succinic acid, fatty    acid, dodecanoic acid, didodecanoic acid, bile acid, cholic acid,    cholesterylhemmisuccinate, a di- or tripeptide, an oligopeptide, a    trisacharide, or a di- or trisaccharide monomer unit of heparin,    heparan sulfate, keratin, keratan sulfate, chondroitin,    chondoitin-6-sulfate, chondroitin-4-sulfate, dermatin, dermatan    sulfate, dextran, or hyaluronic acid; and-   n is the number of lipid moity molecules bound to a molecule of X    wherein n is a number from 1 to 1000.

In one embodiment of this invention, low molecular weightphosphatidylserine (PS)-conjugates are defined hereinabove as thecompounds of formula (II) wherein:

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a mono- or disaccharide, carboxylated disaccharide, mono- or    dicarboxylic acids, a salicylate, salicylic acid, aspirin,    lactobionic acid, maltose, an amino acid, glycine, acetic acid,    butyric acid, dicarboxylic acid, glutaric acid, succinic acid, fatty    acid, dodecanoic acid, didodecanoic acid, bile acid, cholic acid,    cholesterylhemmisuccinate, a di- or tripeptide, an oligopeptide, a    trisaccharide, or a di- or trisaccharide monomer unit of heparin,    heparan sulfate, keratin, keratan sulfate, chondroitin,    chondoitin-6-sulfate, chondroitin-4-sulfate, dermatin, dermatan    sulfate, dextran, or hyaluronic acid; and-   n is the number of lipid moiety molecules bound to a molecule of X    wherein n is a number from 1 to 1000.

In one embodiment of this invention, Phosphatidylcholine (PC),Phosphatidylinositol (PI), and Phosphatidylglycerol (PG) conjugates arehereinabove defined as the compounds of formula (III) wherein:

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a mono- or disaccharide, carboxylated disaccharide, mono- or    dicarboxylic acids, a salicylate, salicylic acid, aspirin,    lactobionic acid, maltose, an amino acid, glycine, acetic acid,    butyric acid, dicarboxylic acid, glutaric acid, succinic acid, fatty    acid, dodecanoic acid, didodecanoic acid, bile acid, cholic acid,    cholesterylhemmisuccinate, a di- or tripeptide, an oligopeptide, a    trisaccharide, or a di- or trisaccharide monomer unit of heparin,    heparan sulfate, keratin, keratan sulfate, chondroitin,    chondoitin-6-sulfate, chondroitin-4-sulfate, dermatin, dextran    sulfate, dextran, or hyaluronic acid; and-   n is the number of lipid moiety molecules bound to a molecule of X    wherein n is a number from 1 to 1000.

Examples of suitable divalent groups forming the optional bridging groupY are straight- or branched -chain alkylene, e.g., of 2 or more,preferably 4 to 18 carbon atoms, —CO-alkylene-CO, —NH-alkylene-NH—,—CO-alkylene-NH—, cycloalkylene, wherein alkylene in each instance, isstraight or branched chain and contains 2 or more, preferably 2 to 18carbon atoms in the chain, —(—O—CH(CH₃)CH₂—)_(x)— wherein x is aninteger of 1 or more.

In another embodiment, in addition to the traditional phospholipidstructure, related derivatives for use in this invention arephospholipids modified at the C1 or C2 position to contain an ether oralkyl bond instead of an ester bond. These derivatives are exemplifiedhereinabove by the general formulae (VIII) and (IX) wherein:

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, ethanolanine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a mono- or disaccharide, carboxylated disaccharide, mono- or    dicarboxylic acids, a salicylate, salicylic acid, aspirin,    lactobionic acid, maltose, an amino acid, glycine, acetic acid,    butyric acid, dicarboxylic acid, glutaric acid, succinic acid, fatty    acid, dodecanoic acid, didodecanoic acid, bile acid, cholic acid,    cholesterylhemmisuccinate, a di- or tripeptide, an oligopeptide, a    trisaccharide, or a di- or trisaccharide monomer unit of heparin,    heparan sulfate, keratin, keratan sulfate, chondroitin,    chondoitin-6-sulfate, chondroitin-4-sulfate, dermatin, dermatan    sulfate, dextran, or hyaluronic acid; and-   n is the number of lipid moity molecules bound to a molecule of X    wherein n is a number from 1 to 1000.

In another embodiment, related low molecular weight derivatives for usein this invention are exemplified hereinabove by the general formulae(X), (XI) and (XII) wherein:

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a mono- or disaccharide, carboxylated disaccharide, mono- or    dicarboxylic acids, a salicylate, salicylic acid, aspirin,    lactobionic acid, maltose, an amino acid, glycine, acetic acid,    butyric acid, dicarboxylic acid, glutaric acid, succinic acid, fatty    acid, dodecanoic acid, didodecanoic acid, bile acid, cholic acid,    cholesterylhemmisuccinate, a di- or tripeptide, an oligopeptide, a    trisaccharide, or a di- or trisaccharide monomer unit of heparin,    heparan sulfate, keratin, keratan sulfate, chondroitin,    chondoitin-6-sulfate, chondroitin-4-sulfate, dermiatin, dermatan    sulfate, dextran, or hyaluronic acid; and-   n is the number of lipid moiety molecules bound to a molecule of X    wherein n is a number from 1 to 1000.

In another embodiment, related low molecular weight derivatives for usein this invention are exemplified hereinabove by the general formulae(XIII) wherein:

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a mono- or disaccharide, carboxylated disaccharide, mono- or    dicarboxylic acids, a salicylate, salicylic acid, aspirin,    lactobionic acid, maltose, an amino acid, glycine, acetic acid,    butyric acid, dicarboxylic acid, glutaric acid, succinic acid, fatty    acid, dodecanoic acid, didodecanoic acid, bile acid, cholic acid,    cholesterylhemmisuccinate, a di- or tripeptide, an oligopeptide, a    trisaccharide, or a di- or trisaccharide monomer unit of heparin,    heparan sulfate, keratin, keratan sulfate, chondroitin,    chondoitin-6-sulfate, chondroitin-4-sulfate, dermatin, dermatan    sulfate, dextran, or hyaluronic acid; and-   n is the number of lipid moiety molecules bound to a molecule of X    wherein n is a number from 1 to 1000.

In another embodiment, related low molecular weight derivativesaccording to the invention may be exemplified herein by any of thegeneral formulae (A), (I)-(XXI) wherein:

In one embodiment of the invention, x is covalently conjugated to alipid. In another embodiment, x is covalently conjugated to a lipid viaan amide bond. In another embodiment, x is covalently conjugated to alipid via an esteric bond. In another embodiment, the lipid isphosphatidylethanolamine. In another embodiment, the GAG may be, interalia, chondroitin sulfate. In another embodiment, the conjugate isbiodegradable.

In one embodiment, the invention provides glycosaminoglycans (GAG)compound covalently conjugated to a lipid to obtain a compound havingpreferred therapeutic properties. In another embodiment, the GAGcompound is covalently conjugated to a lipid via an amide bond. Inanother embodiment, the GAG compound is covalently conjugated to a lipidvia an esteric bond. In another embodiment, the lipid may be, interalia, phosphatidylethanolamine. In another embodiment, the GAG may be,inter alia, chondroitin sulfate. In another embodiment, the conjugate isbiodegradable.

Cell surface GAG play a key role in protecting cells from diversedamaging agents and processes, such as reactive oxygen species and freeradicals, endotoxins, cytokines, invasion promoting enzymes, and agentsthat induce and/or facilitate degradation of extracellular matrix andbasal membrane, cell invasiveness, white cell extravasation andinfiltration, chemotaxis, and others. In addition, cell surface GAGprotect cells from bacterial, viral and parasite infection, and theirstripping exposes the cell to interaction and subsequent internalizationof the microorganism. Enrichment of cell surface GAG would thus assistin protection of the cell from injurious processes. Thus, In oneembodiment of the invention, PLA2 inhibitos were conjugated to GAGs orGAG-mimicking molecules. In another embodiment, these Lipid-conjugates,provides wide-range protection from diverse injurious processes, and areeffective in amelioration of diseases that requires cell protection frominjurous biochemical medistors.

In another embodiment, GAG-mimicking molecule may be, inter alia, anegatively charged molecule. In another embodiment, GAG-mimickingmolecule may be, inter alia, a salicilate derivative. In anotherembodiment, GAG-mimicking molecule may be, inter alia, a dicarboxylicacid.

Preparation of Compounds

The preparation of some high molecular weight Lipid-conjugates is thesubject of U.S. Pat. No. 5,064,817, which is incorporated herein byreference. These synthetic methods are reiterated below and areconsidered to be applicable as well to the preparation of low molecular,i.e. Lipid-conjugates comprising monomers and dimers as the conjugatedmoiety, with modifications in the procedure as readily evident to oneskilled in the art.

When the starting compound chosen for the conjugated moiety has asubstituent which is or can be rendered reactive to a substituent on thestarting Lipid compound, the conjugated carrier moiety may be linkeddirectly to lipid molecule(s) to produce the a Lipid-conjugate. When itdoes not, a bifunctional linking starting material can be used to linkthe two molecules indirectly.

Lipid-conjugates are prepared by linking a polar conjugate, e.g., amonomer or polymer, directly or indirectly to a PL moiety according tothe general reaction schemes delineated in U.S. Pat. No. 5,064,817.

For example, with acylated PE used as precursor for the PE conjugate,various lengths of dicarboxylic acids can be used as spacers. Theseacids can be linked to natural, semi-synthetic or synthetic PE.

For example, PE can be linked to aminodextran indirectly as delineatedin U.S. Pat. No. 5,064,817.

Polymers with carboxylic groups, such as polyamino acids, carboxymethylcellulose or polymers to which fatty acids have been linked, can belinked directly to PE according to the scheme delineated in U.S. Pat.No. 5,064,817.

It is to be understood that these examples are given by way ofillustration only and are not to be construed as limiting the inventioneither in spirit of in scope, as many modifications both in reagents andmethods could be possible to those skilled in the art Based on the widespectrum of pharmacological properties exhibited by Lipid-conjugates, itis likely that compounds covered by Formula I-XXI, in addition to thoseexplicitly described above, have the same valuable biological activitiesdemonstrate to be useful in the methods of treating disease describedbelow.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (A):

wherein

-   L is a lipid or a phospholipid;-   Z is either nothing, ethanolainine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein X is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between L, Z, Y and X is either an amide or an    esteric bond, including, inter alia, the steps of:    -   conjugating L to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, L is conjugated directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, L is conjugated directly to X,    -   thereby preparing a compound represented by the structure of the        general formula (A).

In another embodiment, the invention provides a process for thepreparation of a compound represented by the structure of the generalformula (I):

wherein

-   -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is either a physiologically acceptable monomer, dimer,        oligomer or a physiologically acceptable polymer, wherein X is a        glycosaminoglycan; and    -   n is a number from 1 to 1,000;        wherein if Y is nothing the phosphatidylethanolamine is directly        linked to X via an amide bond and if Y is a spacer, the spacer        is directly linked to X via an amide or an esteric bond and to        the phosphatidylethanolamine via an amide bond, including, inter        alia, the steps of:    -   conjugating the phosphatidylethanolamine to Y; and    -   conjugating Y to X;    -   if Y is nothing, the phosphatidylethanolainine is conjugated        directly to X, thereby preparing a compound represented by the        structure of the general formula (I).

In one embodiment of the invention, the phosphatidylethanolamine is thechemical moiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In another embodiment, the invention provides a process for thepreparation of a compound represented by the structure of the generalformula (II):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein if Y is nothing the phosphatidylserine is directly linked to    X via an amide bond and if Y is a spacer, the spacer is directly    linked to X via an amide or an esteric bond and to the    phosphatidylserine via an amide bond, including, inter alia, the    steps of:    -   conjugating the phosphatidylserine to Y;    -   conjugating Y to X;    -   if Y is nothing, the phosphatidylserine is conjugated directly        to X,    -   thereby preparing a compound represented by the structure of the        general formula (II).

In one embodiment of the invention, the phosphatidylserine is thechemical moiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (III):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phosphatidyl, Z, Y and X is either an    amide or anesteric bond, including, inter alia, the steps of:    -   conjugating the phosphatidyl to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phosphatidyl is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phosphatidyl is conjugated directly        to X,    -   thereby preparing a compound represented by the structure of the        general formula (III).

In one embodiment of the invention, the phosphatidyl may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (IV):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the phospholipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phospholipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phospholipid is conjugated directly        to X,    -   thereby preparing a compound represented by the structure of the        general formula (IV).

In one embodiment of the invention, the phospholipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (V):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the phospholipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phospholipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phospholipid is conjugated directly        to X,    -   thereby preparing a compound represented by the structure of the        general formula (V).

In one embodiment of the invention, the phospholipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (VI):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the phospholipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phospholipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phospholipid is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (VI).

In one embodiment of the invention, the phospholipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (VII):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, inositol, choline, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the phospholipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phospholipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phospholipid is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (VII).

In one embodiment of the invention, the phospholipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula(VIII):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the phospholipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phospholipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phospholipid is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (VIII).

In one embodiment of the invention, the phospholipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (IX):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the phospholipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phospholipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phospholipid is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (IX).

In one embodiment of the invention, the phospholipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (IXa):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the phospholipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phospholipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phospholipid is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (IXa).

In one embodiment of the invention, the phospholipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (IXb):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the phospholipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the phospholipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the phospholipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the phospholipid is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (IXb).

In one embodiment of the invention, the phospholipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (X):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer, or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the ceramide phosphoryl, Z, Y and X is    either an amide or an esteric bond, including, inter alia, the steps    of:    -   conjugating the ceramide phosphoryl to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the ceramide phosphoryl is conjugated        directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the ceramide phosphoryl is conjugated        directly to X, thereby preparing a compound represented by the        structure of the general formula (X).

In one embodiment of the invention, the ceramide phosphoryl may be thechemical moiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (XI):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein if Y is nothing the sphingosyl is directly linked to X via    an amide bond and if Y is a spacer, the spacer is directly linked to    X and to the sphingosyl via an amide bond and to X via an amide or    an esteric bond, including, inter alia, the steps of:    -   conjugating the sphingosyl to Y;    -   conjugating Y to X;    -   wherein if Y is nothing, the sphingosyl is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (XI).

In one embodiment of the invention, the sphingosyl may be the chemicalmoiety represented by the structure of:

wherein R₁ is defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (XII):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   L is ceramide;-   Z is either nothing, ethanolamine, serine, inositol, choline, or    glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the ceramide, Z, Y and X is either an amide    or an esteric bond, including, inter alia, the steps of:    -   conjugating the ceramide to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the ceramide is conjugated directly to        Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the ceramide is conjugated directly to        X, thereby preparing a compound represented by the structure of        the general formula (XII).

In one embodiment of the invention, the ceramide may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula(XIII):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the diglyceryl, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the diglyceryl to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the diglyceryl is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the diglyceryl is conjugated directly to        X, thereby preparing a compound represented by the structure of        the general formula (XIII).

In one embodiment of the invention, the diglyceryl may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (XIV):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the glycerolipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the glycerolipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the glycerolipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the glycerolipid is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (XIV).

In one embodiment of the invention, the glycerolipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (XV):

wherein

-   R₁ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the glycerolipid, Z, Y and X is either an    amide or an esteric bond, including, inter alia, the steps of:    -   conjugating the glycerolipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the glycerolipid is conjugated directly        to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the glycerolipid is conjugated directly        to X, thereby preparing a compound represented by the structure        of the general formula (XV).

In one embodiment of the invention, the glycerolipid may be the chemicalmoiety represented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (XVI):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond, including, inter alia, the steps of:    -   conjugating the lipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the lipid is conjugated directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the lipid is conjugated directly to X,        thereby preparing a compound represented by the structure of the        general formula (XVI).

In one embodiment of the invention, the lipid may be the chemical moietyrepresented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula(XVII):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,    alkyl chain ranging in length from 2 to 30 carbon atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond, including, inter alia, the steps of:    -   conjugating the lipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the lipid is conjugated directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the lipid is conjugated directly to X,        thereby preparing a compound represented by the structure of the        general formula (XVII).

In one embodiment of the invention, the lipid may be the chemical moietyrepresented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula(XVIII):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond, including, inter alia, the steps of:    -   conjugating the lipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the lipid is conjugated directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the lipid is conjugated directly to X,        thereby preparing a compound represented by the structure of the        general formula (XVIII).

In one embodiment of the invention, the lipid may be the chemical moietyrepresented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (XIX):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond, including, inter alia, the steps of:    -   conjugating the lipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the lipid is conjugated directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the lipid is conjugated directly to X,        thereby preparing a compound represented by the structure of the        general formula (XIX).

In one embodiment of the invention, the lipid may be the chemical moietyrepresented by the structure of:

wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (XX):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is anumber from 1 to 1000;    wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond, including, inter alia, the steps of:    -   conjugating the lipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the lipid is conjugated directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the lipid is conjugated directly to X,        thereby preparing a compound represented by the structure of the        general formula (XX).

In one embodiment of the invention, the lipid may be the chemical moietyrepresented by the structure of:

-   -   wherein R₁ and R₂ are defined herein.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of the general formula (XXI):

wherein

-   R₁ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   R₂ is either hydrogen or a linear, saturated, mono-unsaturated, or    poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon    atoms;-   Z is either nothing, choline, phosphate, inositol, or glycerol;-   Y is either nothing or a spacer group ranging in length from 2 to 30    atoms;-   X is a physiologically acceptable monomer, dimer, oligomer or    polymer, wherein x is a glycosaminoglycan; and-   n is a number from 1 to 1000;-   wherein any bond between the lipid, Z, Y and X is either an amide or    an esteric bond, including, inter alia, the steps of:    -   conjugating the lipid to Z;    -   conjugating Z to Y;    -   conjugating Y to X;    -   wherein if Z is nothing, the lipid is conjugated directly to Y,    -   if Y is nothing, Z is conjugated directly to X, and    -   if Y and Z are nothing, the lipid is conjugated directly to X,        thereby preparing a compound represented by the structure of the        general formula (XXI).

In one embodiment of the invention, the lipid may be the chemical moietyrepresented by the structure of:

wherein R₁ and R₂ are defined herein.

In another embodiment, the conjugating according to the invention, maybe performed by eliminating a water molecule, thereby forming amide oresteric bonds. In another embodiment, the conjugating may be performedin the presence of a detergent. In another embodiment, the conjugatingmay be induced by ultrasonic radiation.

In another embodiment, any conjugation process according to theinvention may be performed by eliminating a water molecule, therebyforming amide or esteric bonds. In another embodiment, any conjugationprocess according to the invention may be performed in the presence of adetergent. In another embodiment, any conjugation process according tothe invention may be induced by ultrasonic radiation.

In another embodiment, any compound according to the invention may beprepared by a conjugation process performed by eliminating a watermolecule, thereby forming amide or esteric bonds. In another embodiment,any compound according to the invention may be prepared by a conjugationprocess in the presence of a detergent. In another embodiment, anycompound according to the invention may be prepared by a conjugationprocess induced by ultrasonic radiation.

In one embodiment of the invention, the conjugation of thephosphatidylethanolamine and chondroitin sulfate is performed in thepresence of a detergent. In another embodiment a detergent may be, interalia, DDAB. Of course any other appropriate detergent may be used.

In one embodiment of the invention, the conjugation of thephosphatidylethanolainine and hyaluronic acid is iduced by sonication.

Methods of Treating Disease Based on PL Conjugates

In one embodiment of the invention, the Lipid-conjugates describedherein can be used to treat disease, through exerting at least one oftheir many pharmacological activities, among which are amelioration, orprevention, of tissue injury arising in the course of pathologicaldisease states by stabilizing cell membranes; limiting oxidative damageto cell and blood components; limiting cell proliferation, cellextravasation and (tumor) cell migratory behavior; suppressing immuneresponses; or attenuating physiological reactions to stress, asexpressed in elevated chemokine levels. The medicinal properties ofthese compounds are readily exemplified in using animal models of theparticular disease in which it is desired to use the drug. The patientsto whom the lipid or PL conjugates should be administered are those thatare experiencing symptoms of disease or who are at risk of contractingthe disease or experiencing a recurrent episode or exacerbation of thedisease. The efficacy of these compounds in cellular and animal modelsof disease are described below in The Examples.

The combination of lipids, such as, but not limited tophosphatidylethanolamine and phosphatidylserine, with additional monomeror polymer moieties, is thus a practical route to the production of newdrugs for medical purposes, provided that the resultant chemicalcomposition displays the desired range of pharmacological properties. Inthe cases described herein, the diversity of biological activities andthe effectiveness in disease exhibited by the compounds far exceed theproperties anticipated by use of the starting materials themselves, whenadministered alone or in combination. However, it is likely that the PLconjugate compounds, alone or in combination, will prove to be valuabledrugs when adapted to methods of disease treatment other to thoseconditions specifically described herein.

In one embodiment, the invention provides a method of treating a subjectafflicted with a disease related to chlamydia infection, a disorder ofsmooth muscle cell proliferation, metastatic cancer, obstructiverespiratory disease, colitis, Crohn's disease, or another form ofintestinal mucosal injury, cardiovascular disease, atherosclerosis,central nervous system tissue insult, multiple sclerosis, contactdermatitis, psoriasis, cellular proliferative disorder, sepsis, acuterespiratory distress syndrome, autoimmune disease, hemolysis, HIVinfection, or conjunctivitis.

In one embodiment, the invention provides a method of treating a subjectrequiring anti-oxidant therapy, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject requiring ananti-oxidant therapy.

In one embodiment, the invention provides a method treating a subjectrequiring anti-TNF therapy, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject requiring ananti-TNF therapy.

In one embodiment, the invention provides a method of treating a subjectsuffering from a disorder of smooth muscle cell proliferation,including, inter alia, the step of administering to a subject aneffective amount of a lipid or phospholipid moiety bonded to aphysiologically acceptable monomer, dimer, oligomer, or polymer, therebytreating the subject suffering from a disorder related to smooth musclecell proliferation.

In one embodiment, the invention provides a method of treating a subjectundergoing vascular catheterization, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject undergoingvascular catheterization.

In one embodiment, the invention provides a method of treating a subjectsuffering from metastatic cancer, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering frommetastatic cancer.

In one embodiment, the invention provides a method of treating a subjectsuffering from obstructive respiratory disease, including, inter alia,the step of administering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering fromobstructive respiratory disease.

In one embodiment, the invention provides a method of treating a subjectsuffering from colitis, Crohn's disease, or another form of intestinalmucosal injury, including, inter alia, the step of administering to asubject an effective amount of a lipid or phospholipid moiety bonded toa physiologically acceptable monomer, dimer, oligomer, or polymer,thereby treating the subject suffering from intestinal mucosal injury,including colitis or Crolin's disease.

In one embodiment, the invention provides a method of treating a subjectsuffering from cardiovascular disease, including, inter alia, the stepof administering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering froma cardiovascular disease.

The present invention provides a method of treating a subject sufferingfrom atherosclerosis, including, inter alia, the step of administeringto a subject an effective amount of a lipid or phospholipid moietybonded to a physiologically acceptable monomer, dimer, oligomer, orpolymer, thereby treating the subject suffering from atherosclerosis.

In one embodiment, the invention provides a method of treating a subjectsuffering from central nervous system tissue insult, including, interalia, the step of administering to a subject an effective amount of alipid or phospholipid moiety bonded to a physiologically acceptablemonomer, dimer, oligomer, or polymer, thereby treating the subjectsuffering from a central nervous system insult.

In one embodiment, the invention provides a method of treating a subjectsuffering from multiple sclerosis, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering frommultiple sclerosis.

In one embodiment, the invention provides a method of treating a subjectsuffering from contact dermatitis, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering fromcontact dermatitis.

In one embodiment, the invention provides a of treating a subjectsuffering from psoriasis, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering frompsoriasis.

In one embodiment, the invention provides a method of treating a subjectsuffering from a cellular proliferative disorder, including, inter alia,the step of administering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering froma cellular proliferative disorder.

In one embodiment, the invention provides a method of treating a subjectsuffering from sepsis, including, inter alia, the step of administeringto a subject an effective amount of a lipid or phospholipid moietybonded to a physiologically acceptable monomer, dimer, oligomer, orpolymer, thereby treating the subject suffering from sepsis.

In one embodiment, the invention provides a method of treating a subjectsuffering from ARDS, comprising the steps of administering to a subjectan effective amount of a lipid or phospholipid moiety bonded to aphysiologically acceptable monomer, dimer, oligomer, or polymer, therebytreating the subject suffering from ARDS.

In one embodiment, the invention provides a method of treating a subjectsuffering from autoimmune disease, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering froman autoimmune disease.

In one embodiment, the invention provides a method of treating a subjectsuffering from hemolysis, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering fromhemolysis.

In one embodiment, the invention provides a method of treating a subjectundergoing tissue transplantation or allograft rejection, including,inter alia, the step of administering to a subject an effective amountof a lipid or phospholipid moiety bonded to a physiologically acceptablemonomer, dimer, oligomer, or polymer, thereby treating the subjectundergoing tissue transplantation or allograft rejection.

In one embodiment, the invention provides a method of treating a subjectafflicted with HIV infection, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject afflicted withHIV infection.

In one embodiment, the invention provides a method of treating a subjectafflicted with conjunctivitis, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject afflicted withconjunctivitis.

In one embodiment, the invention provides a method for extracorporealtissue preservation, including, inter alia, the step of adding to atissue preparation or organ an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby extending the viability of thetissue preparation or organ within a donor subject.

In one embodiment, the invention provides a method of treating a subjectafflicted with Chlamydia infection, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject afflictedsuffering from Chlamydia infection.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject requiring an anti-oxidant therapy.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject requiring an anti-TNF therapy.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from a disorder related tosmooth muscle cell proliferation.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject undergoing vascular catheterization.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering form metastatic cancer.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from obstructiverespiratory disease.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from intestinal mucosalinjury, including, inter alia, colitis or Crohn's disease.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from a cardiovasculardisease.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from atherosclerosis.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from central nervous systeminsult.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from multiple sclerosis.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from contact dermatitis.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from psoriasis.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from a cellularproliferative disorder.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from sepsis.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from ARDS.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from an autoimmune disease.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject suffering from hemolysis.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject undergoing tissue transplantation orallograft rejection.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject afflicted with HIV infection.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject afflicted with conjunctivitis.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for extending the viability of the tissue preparation ororgan within a donor subject.

In one embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject afflicted with Chlamydia infection.

In one embodiment of the invention, the treatment requires controllingthe expression production and activity of phospholipase enzymes. Inanother embodiment, the treatment requires controlling the productionand/or action of lipid mediators. In another embodiment, the treatmentrequires amelioration of damage to glycosaminoglycans (GAG) andproteoglycans. In another embodiment, the treatment requires controllingthe production and action of oxidants, oxygen radicals and nitric oxide.In another embodiment, the treatment requires anti-oxidant therapy. Inanother embodiment, the treatment requires anti-endotoxin therapy. Inanother embodiment, the treatment requires controlling the expression,production or action of cytokines, chemokines, adhesion molecules orinterleukines. In another embodiment, the treatment requires protectionof lipoproteins from damaging agents. In another embodiment, thetreatment requires controlling the proliferation of cells. In anotherembodiment, the treatment requires controlling of angiogenesis and organvascularization. In another embodiment, the treatment requiresinhibition of invasion-promoting enzymes. In another embodiment, thetreatment requires controlling of cell invasion. In another embodiment,the invading cells are white blood cells. In another embodiment, theinvading cells are cancer cells. In another embodiment, the treatmentrequires controlling of white cell activation, adhesion orextravasation. In another embodiment, the treatment requiresamelioration of ischemia or reperfusion injury. In another embodiment,the treatment requires inhibition of lymphocyte activation. In anotherembodiment, the treatment requires protection of blood brain barrier. Inanother embodiment, the treatment requires control of neurotransmitterproduction and action. In another embodiment, the treatment requirescontrolling of blood vessel and airway contraction. In anotherembodiment, the treatment requires extracorporeal tissue preservation.

In one embodiment of the invention, the lipid mediator is aglycerolipid. In another embodiment, the lipid mediator is aphospholipid. In another embodiment, the lipid mediator is sphingolipid.In another embodiment, the lipid mediator is a sphingosine. In anotherembodiment, the lipid mediator is ceramide. In another embodiment, thelipid mediator is a fatty acid. In another embodiment, the fatty acid isarachidonic acid. In another embodiment, the lipid mediator is anarachidonic acid-derived eicosanoid. In another embodiment, the lipidmediator is a platelet activating factor (PAF). In another embodiment,the lipid mediator is a lysophospholipid.

In one embodiment of the invention, the damaging agent is aphospholipase. In another embodiment, the damaging agent is a reactiveoxygen species (ROS). In another embodiment, the damaging agent is afree radical. In another embodiment, the damaging agent is alysophospholipid. In another embodiment, the damaging agent is a fattyacid or a derivative thereof. In another embodiment, the damaging agentis hydrogen peroxide. In another embodiment, the damaging agent is aphospholipid. In another embodiment, the damaging agent is an oxidant.In another embodiment, the damaging agent is a cationic protein. Inanother embodiment, the damaging agent is a streptolysin. In anotherembodiment, the damaging agent is a protease. In another embodiment, thedamaging agent is a hemolysin. In another embodiment, the damaging agentis a sialidase.

In one embodiment of the invention, the invasion-promoting enzyme iscollagenase. In another embodiment, the invasion-promoting enzyme ismatrix-metaloproteinase (MMP). In another embodiment, theinvasion-promoting enzyme is heparinase. In another embodiment, theinvasion-promoting enzyme is heparanase. In another embodiment, theinvasion-promoting enzyme is hyaluronidase. In another embodiment, theinvasion-promoting enzyme is gelatinase. In another embodiment, theinvasion-promoting enzyme is chondroitinase. In another embodiment, theinvasion-promoting enzyme is dermatanase. In another embodiment, theinvasion-promoting enzyme is keratanase. In another embodiment, theinvasion-promoting enzyme is protease. In another embodiment, theinvasion-promoting enzyme is lyase. In another embodiment, theinvasion-promoting enzyme is hydrolase. In another embodiment, theinvasion-promoting enzyme is a glycosaminoglycan degrading enzyme. Inanother embodiment, the invasion-promoting enzyme is a proteoglycandegrading enzyme.

In one embodiment of the invention, the physiologically acceptablemonomer is salicylate. In another embodiment, the physiologicallyacceptable monomer is salicylic acid. In another embodiment, thephysiologically acceptable monomer is aspirin. In another embodiment,the physiologically acceptable monomer is a monosaccharide. In anotherembodiment, the physiologically acceptable monomer is lactobionic acid.In another embodiment, the physiologically acceptable monomer isglucoronic acid. In another embodiment, the physiologically acceptablemonomer is maltose. In another embodiment, the physiologicallyacceptable monomer is an amino acid. In another embodiment, thephysiologically acceptable monomer is glycine. In another embodiment,the physiologically acceptable monomer is a carboxylic acid. In anotherembodiment, the physiologically acceptable monomer is an acetic acid. Inanother embodiment, the physiologically acceptable monomer is a butyricacid. In another embodiment, the physiologically acceptable monomer is adicarboxylic acid. In another embodiment, the physiologically acceptablemonomer is a glutaric acid. In another embodiment, the physiologicallyacceptable monomer is succinic acid. In another embodiment, thephysiologically acceptable monomer is a fatty acid. In anotherembodiment, the physiologically acceptable monomer is dodecanoic acid.In another embodiment, the physiologically acceptable monomer isdidodecanoic acid. In another embodiment, the physiologically acceptablemonomer is bile acid. In another embodiment, the physiologicallyacceptable monomer is cholic acid. In another embodiment, thephysiologically acceptable monomer is cholesterylhemmisuccinate.

In one embodiment of the invention, the physiologically acceptable dimeror oligomer is physiologically acceptable dimer or oligomer is adipeptide. In another embodiment, the physiologically acceptable dimeror oligomer is a disaccharide. In another embodiment, thephysiologically acceptable dimer or oligomer is a trisaccharide. Inanother embodiment, the physiologically acceptable dimer or oligomer isan oligosaccharide. In another embodiment, the physiologicallyacceptable dimer or oligomer is an oligopeptide. In another embodiment,the physiologically acceptable dimer or oligomer is a di- ortrisaccharide monomer unit of glycosaminoglcans. In another embodiment,the physiologically acceptable dimer or oligomer is hyaluronic acid. Inanother embodiment, the physiologically acceptable dimer or oligomer isheparin. In another embodiment, the physiologically acceptable dimer oroligomer is heparan sulfate. In another embodiment, the physiologicallyacceptable dimer or oligomer is keratin. In another embodiment, thephysiologically acceptable dimer or oligomer is keratan sulfate. Inanother embodiment, the physiologically acceptable dimer or oligomer ischondroitin. In another embodiment, the chondroitin is chondoitinsulfate. In another embodiment, the chondroitin is chondoitin-4-sulfate.In another embodiment, the chondroitin is chondoitin-6-sulfate. Inanother embodiment, the physiologically acceptable dimer or oligomer isdermatin. In another embodiment, the physiologically acceptable dimer oroligomer is dermatan sulfate. In another embodiment, the physiologicallyacceptable dimer or oligomer is dextran. In another embodiment, thephysiologically acceptable dimer or oligomer is polygeline(‘Haemaccel’). In another embodiment, the physiologically acceptabledimer or oligomer is alginate, In another embodiment, thephysiologically acceptable dimer or oligomer is hydroxyethyl starch(Hetastarch). In another embodiment, the physiologically acceptabledimer or oligomer is ethylene glycol. In another embodiment, thephysiologically acceptable dimer or oligomer is carboxylated ethyleneglycol.

In one embodiment of the invention, the physiologically acceptablepolymer is a glycosaminoglycan. In another embodiment, thephysiologically acceptable polymer is hyaluronic acid. In anotherembodiment, the physiologically acceptable polymer is heparin. Inanother embodiment, the physiologically acceptable polymer is heparansulfate. In another embodiment, the physiologically acceptable polymeris chondroitin. In another embodiment, the chondroitin ischondoitin-4-sulfate. In another embodiment, the chondroitin ischondoitin-6-sulfate. In another embodiment, the physiologicallyacceptable polymer is keratin. In another embodiment, thephysiologically acceptable polymer is keratan sulfate. In anotherembodiment, the physiologically acceptable polymer is dermatin. Inanother embodiment, the physiologically acceptable polymer is dermatansulfate. In another embodiment, the physiologically acceptable polymeris carboxymethylcellulose. In another embodiment, the physiologicallyacceptable polymer is dextran. In another embodiment, thephysiologically acceptable polymer is polygeline (‘Haemaccel’). Inanother embodiment, the physiologically acceptable polymer is alginate.In another embodiment, the physiologically acceptable polymer ishydroxyethyl starch (‘Hetastarch’). In another embodiment, thephysiologically acceptable polymer is polyethylene glycol. In anotherembodiment, the physiologically acceptable polymer is polycarboxylatedpolyethylene glycol.

In one embodiment of the invention, the lipid or phospholipid moiety isphosphatidic acid. In another embodiment, lipid or phospholipid moietyis an acyl glycerol. In another embodiment, lipid or phospholipid moietyis monoacylglycerol. In another embodiment, lipid or phospholipid moietyis diacylglycerol. In another embodiment, lipid or phospholipid moietyis triacylglycerol. In another embodiment, lipid or phospholipid moietyis sphingosine. In another embodiment, lipid or phospholipid moiety issphingomyelin. In another embodiment, lipid or phospholipid moiety isceramide. In another embodiment, lipid or phospholipid moiety isphosphatidylethanolamine. In another embodiment, lipid or phospholipidmoiety is phosphatidylserine. In another embodiment, lipid orphospholipid moiety is phosphatidylcholine. In another embodiment, lipidor phospholipid moiety is phosphatidylinositol. In another embodiment,lipid or phospholipid moiety is phosphatidylglycerol. In anotherembodiment, lipid or phospholipid moiety is an ether or alkylphospholipid derivative thereof.

In one embodiment, the invention provides a method of treating a subjectafflicted with a disease, wherein the treatment of the disease requirescontrolling phospholipase A2 activities; controlling the productionand/or action of lipid mediators, such as eicosanoids, plateletactivating factor (PAF) and lyso-phospholipids; amelioration of damageto cell surface glycosaminoglycans (GAG) and proteoglycans; controllingthe production of oxygen radicals and nitric oxide; protection of cells,tissues, and plasma lipoproteins from damaging agents, such as reactiveoxygen species (ROS) and phospholipases; anti-oxidant therapy;anti-endotoxin therapy; controlling of cytokine, chemokine andinterleukine production; controlling the proliferation of cells,including smooth muscle cells, endothelial cells and skin fibroblasts;controlling of angiogenesis and organ vascularization; inhibition ofinvasion-promoting enzymes, such as collagenase, heparinase, heparanaseand hyaluronidase; controlling of cell invasion; controlling of whitecell activation, adhesion and extravasation; amelioration of ischemiareperfusion injury, inhibition of lymphocyte activation; controlling ofblood vessel and airway contraction; protection of blood brain barrier;controlling of neurotransmitter (e.g., dopamine) production and action(e.g., acethylcholine); extracorporeal tissue preservation or anycombination thereof.

In one embodiment of the invention, the term “controlling” refers toinhibiting the production and action of the above mentioned factors inorder to maintain their activity at the normal basal level and suppresstheir activation in pathological conditions.

In one embodiment of the invention, the physiologically acceptablemonomer is either a salicylate, salicylic acid, aspirin, amonosaccharide, lactobionic acid, maltose, an amino acid, glycine,carboxylic acid, acetic acid, butyric acid, dicarboxylic acid, glutaricacid, succinic acid, fatty acid, dodecanoic acid, didodecanoic acid,bile acid, cholic acid, cholesterylhemmisuccinate; or wherein thephysiologically acceptable dimer or oligomer is a dipeptide, adisaccharide, a trisaccharide, an oligopeptide, or a di- ortrisaccharide monomer unit of heparin, heparan sulfate, keratin, keratansulfate, chondroitin, chondoitin-6-sulfate, chondroitin-4-sulfate,dermatin, dermatan sulfate, dextran, or hyaluronic acid; or wherein thephysiologically acceptable polymer is a glycosaminoglycan, polygelin(‘haemaccel’), alginate, hydroxyethyl starch (hetastarch), polyethyleneglycol, polycarboxylated polyethylene glycol, chondroitin-6-sulfate,chondroitin-4-sulfate, keratin, keratin sulfate, heparan sulfate,dermatin, dermatan sulfate, carboxymethylcellulose, heparin, dextran, orhyaluronic acid.

In one embodiment of the invention, the lipid moiety is eitherphosphatidic acid, an acyl glycerol, monoacylglycerol, diacylglycerol,triacylglycerol, sphingosine, sphingomyelin, chondroitin-4-sulphate,chondroitin-6-sulphate, ceramide, phosphatidylethanolamine,phosphatidylserine, phosphatidylcholine, phosphatidylinositol, orphosphatidylglycerol, or an ether or alkyl phospholipid derivativethereof, and the physiologically acceptable monomer or polymer moiety iseither aspirin, lactobionic acid, maltose, glutaric acid, polyethyleneglycol, carboxymethylcellulose, heparin, dextran, hemacell, hetastarch,or hyaluronic acid.

In one embodiment, the present invention provides for use of a lipidmoiety bonded to a physiologically acceptable monomer, dimer, oligomer,or polymer, in the preparation of a pharmaceutical composition fortreating a subject afflicted with obstructive respiratory disease,colitis, Crohn's disease, central nervous system insult, multiplesclerosis, contact dermatitis, psoriasis, cardiovascular disease,including prophylaxis for invasive procedures, invasive cellularproliferative disorders, anti-oxidant therapy, hemolytic syndromes,sepsis, acute respiratory distress syndrome, tissue transplant rejectionsyndromes, autoimmune disease, viral infection, and hypersensitivityconjunctivitis.

In one embodiment, the present invention provides use of apharmaceutical composition according to the present invention fortreating a subject afflicted with obstructive respiratory disease,colitis, Crohn's disease, central nervous system insult, multiplesclerosis, contact dermatitis, psoriasis, cardiovascular disease,including prophylaxis for invasive procedures, invasive cellularproliferative disorders, anti-oxidant therapy, hemolytic syndromes,sepsis, acute respiratory distress syndrome, tissue transplant rejectionsyndromes, autoimmune disease, viral infection, or hypersensitivityconjunctivitis, wherein the composition is prepared for administrationby topical, oral, nasal, aerosol, intravenous, intraocular,intra-arterial, subcutaneous, or suppository routes.

In one embodiment, the invention provides a method of treating a subjectsuffering from an intestinal disease, including, inter alia, the step ofadministering to a subject an effective amount of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, thereby treating the subject suffering froman intestinal disease.

In another embodiment, the invention provides a use of a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer, in the preparation of a pharmaceuticalcomposition for treating a subject afflicted with an intestinal disease.

In one embodiment, the invention provides a method of treating a subjectsuffering from a disease involving the production and/or action of lipidmediators and/or impairment of glycosaminoglycan (GAG) functioning.

In another embodiment, the invention provides a pharmaceuticalcomposition for treating a subject suffering from an intestinal disease,including, inter alia, a lipid or phospholipid moiety bonded to aphysiologically acceptable monomer, dimer, oligomer, or polymer; and apharmaceutically acceptable carrier or excipient.

In one embodiment, the intestinal disease may be, inter alia, a diseaseinvolving the production and/or action of lipid mediators and/orimpairment of glycosaminoglycan (GAG) functioning.

In one embodiment of the invention, the intestinal disease may be, interalia, Crohn's disease, ulcerative colitis, immuno-inflammatoryintestinal injury, drug-induced enteropathy, ischemia-induced intestinalinjury or any combination thereof.

In one embodiment of the invention, the physiologically acceptablemonomer may be, inter alia, a salicylate, salicylic acid, aspirin, amonosaccharide, lactobionic acid, glucoronic acid, maltose, amino acid,glycine, carboxylic acid, acetic acid, butyric acid, dicarboxylic acid,glutaric acid, succinic acid, fatty acid, dodecanoic acid, didodecanoicacid, bile acid, cholic acid, cholesterylhemmisuccinate, or wherein thephysiologically acceptable dimer or oligomer may be, inter alia, adipeptide, a disaccharide, a trisaccharide, an oligosaccharide, anoligopeptide, or a di- or trisaccharide monomer unit ofglycosaminoglcans, hyaluronic acid, heparin, heparan sulfate, keratin,keratan sulfate, chondroitin, chondroitin sulfate,chondroitin-4-sulfate, chondoitin-6-sulfate, dermatin, dermatan sulfate,dextran, polygeline, alginate, hydroxyethyl starch, ethylene glycol, orcarboxylated ethylene glycol, or wherein the physiologically acceptablepolymer may be, inter alia, a glycosaminoglycan, hyaluronic acid,heparin, heparan sulfate, chondroitin, chondroitin sulfate, keratin,keratan sulfate, dermatin, dermatan sulfate, carboxymethylcellulose,dextran, polygeline, alginate, hydroxyethyl starch, polyethylene glycolor polycarboxylated polyethylene glycol.

In another embodiment, the physiologically acceptable polymer may be,inter alia, hyaluronic acid.

In another embodiment, the physiologically acceptable polymer may be,inter alia, chondroitin sulfate.

In one embodiment of the invention, the lipid or phospholipid moiety maybe, inter alia, phosphatidic acid, an acyl glycerol, monoacylglycerol,diacylglycerol, triacylglycerol, sphingosine, sphingomyelin, ceramide,phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine,phosphatidylinositol, phosphatidylglycerol, or an ether or alkylphospholipid derivative thereof.

In another embodiment, the phospholipid moiety may be, inter alia,phosphatidylethanolamine.

Dosages and Routes of Administration

The methods of this invention can be adapted to use of the therapeuticcompositions comprising Lipid-conjugates in admixture with conventionalexcipients, i.e. pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral, enteral (e.g., oral) ortopical application which do not deleteriously react with the activecompounds. Suitable pharmaceutically acceptable carriers include but arenot limited to water, salt solutions, alcohols, gum arabic, vegetableoils, benzyl alcohols, polyethylene glycols, gelatine, carbohydratessuch as lactose, amylose or starch, magnesium stearate, talc, silicicacid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronicacid, collagen, perfume oil, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, flavoring and/or aromatic substances andthe like which do not deleteriously react with the active compounds.They can also be combined where desired with other active agents, e.g.,vitamins.

In one embodiment, the invention provides a pharmaceutical compositionfor treating a subject suffering from sepsis, comprising a lipid orphospholipid moiety bonded to a physiologically acceptable monomer,dimer, oligomer, or polymer; and a pharmaceutically acceptable carrieror excipient.

While the examples provided herein describe use of the PL conjugates insubcutaneous, intraperitoneal or topical administration the successdescribed affords good evidence to suppose that other routes ofadministration, or combinations with other pharmaceutical preparations,would be at least as successful. The route of administration (e.g.,topical, parenteral, enteral, intravenous, vaginal, inhalation, nasalaspiration (spray), supository or oral) and the dosage regimen will bedetermined by skilled clinicians, based on factors such as exact natureof the condition being treated, the severity of the condition, the ageand general physical condition of the patient, and so on.

In general, the doses utilized for the above described purposes willvary, but will be in an effective amount to exert the desiredanti-disease effect. As used herein, the term “pharmaceuticallyeffective amount” refers to an amount of a compound of formulae I-XXIwhich will produce the desired alleviation in symptoms or signs ofdisease in a patient. The doses utilized for any of the above-describedpurposes will generally be from 1 to about 1000 milligrams per kilogramof body weight (mg/kg), administered one to four times per day, or bycontinuous IV infusion. When the compositions are dosed topically, theywill generally be in a concentration range of from 0.1 to about 10% w/v,administered 1-4 times per day.

As used herein, the term “pharmaceutically acceptable carrier” refers toany formulation which is safe, and provides the appropriate delivery forthe desired route of administration of an effective amount of at leastone compound of the present invention. As such, all of theabove-described formulations of the present invention are herebyreferred to as “pharmaceutically acceptable carriers.” This term refersto as well the use of buffered formulations wherein the pH is maintainedat a particular desired value, ranging from pH 4.0 to pH 9.0, inaccordance with the stability of the compounds and route ofadministration.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Ampoulesare convenient unit dosages.

For application by inhalation, particularly for treatment of airwayobstruction or congestion, solutions or suspensions of the compoundsmixed and aerosolized or nebulized in the presence of the appropriatecarrier suitable.

For topical application, particularly for the treatment of skin diseasessuch as contact dermatitis or psoriasis, admixture of the compounds withconventional creams or delayed release patches is acceptable.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules. A syrup, elixir, or the likecan be used when a sweetened vehicle is employed. When indicated,suppositories or enema formulations may be the recommended route ofadministration.

Sustained or directed release compositions can be formulated, e.g.,liposomes or those wherein the active compound is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc. It is also possible to freeze-dry the newcompounds and use the lyophilisates obtained, for example, for thepreparation of products for injection.

Thus, the present invention provides for use of the Lipid-conjugates invarious dosage forms suitable for aerosol, rectal, vaginal,conjunctival, intravenous, intra-arterial, and sublingual routes ofadministration.

It will be appreciated that the actual preferred amounts of activecompound in a specific case will vary according to the specific compoundbeing utilized, the particular compositions formulated, the mode ofapplication, and the particular situs and organism being treated.Dosages for a given host can be determined using conventionalconsiderations, e.g., by customary comparison of the differentialactivities of the subject compounds and of a known agent, e.g., by meansof an appropriate, conventional pharmacological protocol.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

EXAMPLES

The main abbreviations used in the examples below are:

-   HA=hyaluronic acid-   HYPE=dipalmitoyl-phosphatidyl-ethanolamine (PE) conjugated to HA    (also referred to as HyPE, HyalPE)-   CSA=chondroitin sulfate A-   CSAPE=PE conjugated to CSA (also referred to as CsAPE, CsaPE)-   CMC=carboxymethyl cellulose-   CMPE=PE conjugated to CMC-   HEPPE=PE conjugated to heparin (also referred to as HepPE, HePPE)-   DEXPE=PE conjugated to dextran-   AsPE=PE conjugates to aspirin-   HemPE=PE conjugated to Polygeline (haemaccel)-   HyDMPE=dimyristoyl PE linked to HA.

EXAMPLE 1 Obstructive Respiratory Disease

The Lipid-conjugates are effective in the treatment of obstructiverespiratory disease. This is demonstrated for asthma in the Experiments1-8 below. In asthma, the impeded airflow is due to airway obstructionwhich is the result of constriction and obstruction of luminal vesselsof the lungs. One widely-accepted experimental system to investigateairway constriction is to induce smooth muscle preparations, isolatedfrom airways, to contract in the absence and presence of the drug.Another widely-accepted test of anti-asthma drug action is to use liveanimals which have asthma. This disease is present in animals which havebeen sensitized to an antigen and which can be monitored forexacerbation and recovery from asthmatic breathing using a bodyplethysmography.

In Experiments 1.1-1.3, the muscle preparation (tracheal rings) wasisolated from rats and in Experiment 1.4-1.5 from guinea pigs. Musclecontraction is measured by attachment of the muscle to a pressuretransducer, which works much like a spring. Induction of contractionoccurs when astlimatogenic substances are administered such asendothelin-1 (ET) an acetylcholine (AcCh).

Experiment 1.1: Isolated rat tracheal rings (in a linear array) werebathed in Krebs-Hanselet buffer (pH=7.4), and linked to a tensiontransducer. ET-1 was added to a final concentration as indicated, andthe tracheal ring contraction was determined by the change in the forceapplied to the tension transducer (FIG. 1.1A). Subsequently, the highestET concentration was used in testing the Lipid-conjugates to inhibit thesmooth muscle contraction. In this experiment (FIG. 1.1B), rat trachearings were incubated with the Lipid-conjugate HyPE at the indicatedconcentration for 1 hr. ET-1 was then added to a final concentration of1 μM and the ring contraction was determined as in Experiment 1.1A. Eachdatum is mean±S.D. of four separate experiments (4 rats).

Experiment 1.2: Rat trachea rings were incubated with 3 μM HYPE orhyaluronic acid (HA) alone, for 1 hr. ET-1 was then added to a finalconcentration of 1 μM (empty bars) or 10 μM (full bars) and the trachealring contraction was determined as in Experiment 1.1 (FIG. 1.2).

Experiment 1.3: The same as Experiment 1.2, but the tracheal ringcontraction was induced by 10 μM Acetyl Choline (AcCh), as shown in FIG.1.3.

Experiment 1.4: Guinea pig tracheal rings (in a linear array), immersedin a ringer bath, were connected to an apparatus measuring the length ofthe ring chain. CMPE or HEPPE was added to the bath 1 h prior to thestimulation of contraction by either Crotalus atrox (type II) enzyme orendothelin-1 as indicated (Table 1.1). TABLE 1.1 Inhibition of TrachealRing Contraction by CMPE and HEPPE Stimulant Lipid-conjugate %Inhibition Phospholipase (0.5 μ/ml) CMPE (10 μM)  100 ± 0.3  (crotalusatrox type II) Histamine (20 μM) CMPE (10 μM)  69 ± 0.1 Histamine (20μM) HEPPE (15 μM)   56 ± 0.05 Endothelin-1 (100 nM) CMPE (10 μM)  92 ±1.1

Experiment 1.5: Guinea pig tracheal rings were incubated with or withoutCMPE for 30 minutes prior to stimulation. The medium was collected after30 minutes and PGE₂ and TXB₂ were determined by radioimmunoassay (Table1.2). (n.d.=below limit of detection.) TABLE 1.2 Inhibition of TrachealTissue PGE₂ and TBX₂ Production by CMPE PGE₂ TXB₂ Stimulant CMPE (ng/ml)(ng/ml) Hitsamine (40 μM) — 5.1 5.6 Histamine (40 μM) 10 μM n.d. 1.75

Experiments 1.6-1.7 demonstrate the ability of Lipid-conjugates to exerttheir pharmacological effect in live animals. In this case, the ratswere sensitized by injection with ovalbumin and then tested forasthmatic disease upon re-exposure to the antigen through therespiratory route. For these experiments, asthma was induced in BrownNorway (BN) rats by subcutaneous (S.C.) injection of ovalbumin (OA) withaluminum hydroxide and intraperitoneal (I.P.) injection of heat-killedBordatella Pertussis on day 1. Bronchoconstriction (challenge) wasinduced on days 14, 16 and 18 by aerosolic administration of OA.Pulmonary functions were tested with each rat in a body-box on day 18, 5min. after challenge.

Penh, (pulmonary airflow obstruction in conscious rats) was determinedusing the method of Harnelmann, et al. Unrestrained conscious rats wereplaced in a whole-body plethysmograph (Buxco Electronics Inc., Troy, NewYork, USA) connected to a preamplifier (model MAX2270, BuxcoElectronics). Analog signals from the amplifier are converted to adigital signal by an AD card (LPM-16 National Instruments Austin, Tex.,USA). Calculations of the enhanced pause (Penh) of each breath are usedas bronclioconstriction measurements. Penh is the result of the formula:Penh=(PEF/PIF)×(Te−Tr)/Tr) where PEF=peak expiratory flow; PIF=peakinspiratory flow; Te=expiratory time; Tr=relaxation time.

Experiment 1.6: Treatment with Lipid-conjugates, dissolved in PBS, orthe vehicle (control) was performed by subcutaneous (S.C.) injection (10mg/100 g body weight) at 24 and 1 hour prior to challenge with OA (FIG.1.4). Each datum is mean ±SEM for 5 rats. *,**p<0.005.

Experiment 1.7: Aerosolic administration of Lipid-conjugates: In eachgroup, 5 rats in a 20 L cage, inhaled the aerosolic preparation of HYPEfor 5 min, one day and 1 hour prior to challenge (FIG. 1.5). Each datumis mean ±SEM for 5 rats. *p<0.01.

Clearly, FIGS. 1.4 and 1.5 demonstrate that the Lipid-conjugates areeffective in treating the asthma both when administered subcutaneously(FIG. 1.4) and as inhaled aerosol. (FIG. 1.5).

Experiment 1.8: The in vivo effects of the Lipid-conjugates aredemonstrated not only by their alleviation of the respiratory distressof sick animals but by histological diagnosis as well (FIG. 1.6).Administration of Lipid-conjugates significantly reduces thephysiological infiltrates of the airway lumen associated with asthmaticdisease, and in this capacity are at least as effective as the standardsteroid-based drug dexamethasone.

These experiments demonstrate that the Lipid-conjugates may be used forthe treatment of obstructive respiratory disease, alleviating airwaynarrowing by a plurality of mechanisms, including inhibition ofcontraction and reduction of airway obstructing infiltrates. Additionalsupport for the utility of the Lipid-conjugates in treating obstructiverespiratory disease is provided by the results of Experiments 7.1-7.3below, demonstrating that the Lipid-conjugates are effective ininhibiting smooth muscle cell proliferation, which is a major cause ofmorbidity in chronic asthma.

EXAMPLE 2 Intestinal Diseases: Crohn's Disease, Ulcerative Colitis,Immuno-Inflammatory Intestinal Injury, Drug-Induced Enteropathy andIschemia-Induced Intestinal Injury

The Lipid-conjugates are effective in the treatment of mucosal layerdamage occuring gastrointestinal (GI) tract disorders. This isdemonstrated in Experiments 2.1-2.4. Ulcerative Colitis and Crohn'sdisease are examples of digestive tract disease in which the mucosalbarrier which lines the gut is damaged. One common model of GI diseaseof this type is the damage to the mucosal lining of the intestines,produced in rodents by high doses of non-steroidal anti-inflammatorydrug (NSAID), such as indomethcin (IND), or toxins such astrinitrobenzene sulfonic acid (TNBS), which induce phenotypes of Crohn'sdisease, or the bowel irritant known as dextran sulfate sodium salt(DSS) (a model of colitis). For experimental protocols see Materials andMethods.

Experiment 2.1: Amelioration of small intestinal injury induced byindomethacin: Indomethacin (Sigma, St Louis, Mo.) a cyclooxygenaseinhibitor used for induction of experimental gut injury in animalmodels, was administered intra-peritoneally (IP, 6 mg in 1 ml of 1%NaHCO₃) to rats weighing (200-250 g) and the development and course ofthe disease were monitored for 5 days, according to our preliminaryexperiments and the previous reports. In the CMPE-treated group, thedrug (20 mg in 1 ml saline) was given I.P. 1 h prior to and 6 h, 24 hand 48 h after indomethacin administration. Control, untreated ratsreceived 1 ml of the vehicle (saline) at the same time points.

Intestinal injury is characterized by permeability to molecules that donot permeate the normal intestine barrier. In the present experiment,intestinal permeability was evaluated by determination of the level ofinulin fluorescein (InFl) in the rat plasma following its oraladministration. It was previously shown [Krimsky et al., 2000] thatalthough InFl does not normally cross the intestine, it readilypermeates the injured intestine, as measured by its appearance in theblood plasma. In the present experiment, InFl was orally given (bygastric intubation) to the healthy (control) and indomethacin-inducedrats on the 3rd day after indomethacin administration, and itsappearance in plasma was determined 3 h later, by measuring thefluorescein fluorescence [Krimsky et al., 2000]. As shown in FIG. 2.1,the intestinal permeation in the CMPE-treated rats was markedly lower(close to the normal range) than in the untreated rats. The retention ofthe intestinal wall barrier intactness, as expressed by the preventionof the fluorescent dye permeation, upon treatment with Lipid-conjugate,demonstrates their protective effect against damage-inducing drug ortoxin on the functional level.

The rats' survival was monitored for 72 h. Table 1 shows that in ratswith indomethacin-induced small intestine injury, the illness isremarkably improved by treatment with Lipid-conjugates, as evidenced bythe marked reduction in the mortality rate among the rats treated withCMPE, compared to the untreated rats. TABLE 2.1 CMPE reduces mortalityof rats with indomethacin-induced small intestinal injury: No. of deadrats {overscore (No. of rats in group)} % mortality Treatment TreatmentPBS CMPE PBS CMPE 2/5 1/5 40 20 2/5 1/5 40 20 3/5 0/5 60  0 TOTAL  7/15 2/15 MEAN ± SEM 46.7 ± 6.7 13.3 ± 6.7 P < 0.025

The surviving rats were sacrificed and examined for macroscopic andhistological damage from the duodenum to the cecum 20 cm of the jejunumwere taken for examination of histological damage. Macroscopic scoringof intestinal damage, from 0 (no damage) to 5 (maximal damage) wasassessed by naked-eye examination of areas of mucosal discoloration,erosion, exudation, ulceration, bowel wall thickening and percentage ofdamaged area. Histological scoring of intestinal damage is the averageof microscopic evaluation of five criteria, ranging from 0 (no damage)to 5 (maximal damage): extent of necrotic area, depth of necrosis, whitecell infiltration intensity and extent, and fibrosis. FIG. 2.2, leftpanel (tissue damage macroscore) and FIG. 2.2, right panel (histologicalscore), demonstrate that treatment with the Lipid-conjugates markedlyameliorated the small intestinal damage.

Experiment 2.2: Amelioration of TNBS-induced colon damage byLipid-conjugates: Colon injury was induced by rectal administration ofTNBS (Sigma, St. Louis, Mo.), 25 mg in 1 ml of 50% EtlOH to untreated orCMPE-treated rats (Hebrew University Sabra rats 200-250 g), after 24 hof food-fasting and the course of the disease was monitored for 2 days.In the Lipid-conjugate-treated group, the rats were injected I.P. with20 mg of CMPE (in 1 ml saline, to obtain about 10 μM in body fluid) atthe following time points: 18 h and 0.5 h prior to, as well as 3 h, 18 hand 36 h after TNBS administration. Control, untreated rats received 1ml of the vehicle (saline) I.P. at the same time points.

Intestinal permeability was tested 12 h after administration of TNBS, byrectal administration of InFl, and determination of its appearance inblood plasma 2 h later. FIG. 2.3 shows that the intestinal permeation inthe CMPE-treated rats was markedly lower (close to the normal range)than in the untreated rats. The preservation of the integrity of theintestinal wall barrier by treatment with Lipid-conjugate, demonstratesthe Lipid-conjugate capacity to ameliorate damage to intestinal mucosa.

Since, as discussed above, activation of phospholipase A2 (PLA2) is animportant determinant of intestinal injury, the effect of treatment withLipid-conjugates, designed to be an PLA2 inhibitors, on PLA2 level inthe colitic rats was also determined. To this end, blood samples weredrawn from the (untreated) coliticrats and the CMPE-treated colitic ratsat different time points after induction of disease with TNBS. Theplasma was separated by centrifugation, and its PLA2 activity wasdetermined by the common method of interacting the enzyme-containingplasma with radioactively-labeled phospholipid membranes, in which thePLA2 activity is expressed by the hydrolysis of the lipid substrate, asmeasured by the resultant free radioactive fatty acid [Krimsky et al.,2003]. As shown in FIG. 2.4, treatment with CMPE reduced the plasma PLA2activity considerably (p=0.011 by Ω-square test for combinedprobability), demonstrating the Lipid-conjugate capacity to control theproduction of injurious lipid mediators.

In addition, it was found that the treatment with the Lipid-conjugateCMPE considerably reduced the myeloperoxidase activity (MPO) in thecolon of colitic rats that had survived; Myeloperoxidase activity intissue homogenate was determined spectroscopically by the common methodof o-dianisidine/H2O2 reaction. The respective MPO activity in theuntreated and the CMPE-treated groups was 19.1±2.6 AND 7.9±1.1 units/mg.Tissue (mean±SEM, n=6, p<0.01).

Monitoring of rat survival through 48 h from induction of disease byadministration of TNBS, revealed that the illness was remarkablyimproved by treatment with Lipid conjugates, as evidenced by the markedreduction in the mortality rate among the CMPE-treated rats, compared tountreated colitis rats, as shown in Table 2.2. TABLE 2.2 CMPE reducesmortality of rats with TNBS-induced colitis. No. of dead rats {overscore(No. of rats in group)} % mortality Treatment Treatment PBS CMPE PBSCMPE 4/8 1/8  50 12.5  4/10 0/10 40 0  7/10 3/10 70 30 5/8 1/8  62 12.5 7/10 4/10 70 40 TOTAL 27/46 9/46 MEAN ± SEM 58.4 ± 5.9 19.0 ± 7.1 P <0.005

The rats that survived the experiment course (48 h) were sacrificed, and10 cm segments of the distal colon were dissected longitudinally,stained for histology (FIG. 2.5), and the area of ulcers (in equivalentcolon sample) was determined by computerized morphometry (FIG. 2.6).*p<0.004 by Mann-Whitney test (n=5).

Experiment 2.3: Amelioration of Colitis induced in mice bydextran-sulfate. Three groups of mice (n=12) were included. Colitis wasinduced by 4% dextran sulfate sodium salt (DSS) (ICN, MW 36,000-44,000)in the drinking water. In Group 1 (DSS), feeding (free drinking) with 4%dextran sulfate sodium (DSS) dissolved in tap water for 7 days followedby plain water for 7 days and treatment was with oral administration(gastric intubation) of solvent (PBS). In Group 2 (DSS+HyPE), feedingwas with 4% dextran sulfate sodium (DSS) dissolved in tap water for 7days followed by plain water for 7 days and treatment with oraladministration (gastric intubation) of HyPE solution in PBS (2×80 μg/gbody weight). Group 3 (healthy control) received plain water for 14days. Drinking water was ad libidum. The body weight was determineddaily (control body weight on the first day of the experiment beforetreatment was started; final body weight on the day of sacrifice).Dextran-treatment was continued until the mean decrease in body weightof the dextran/solvent containing dextran was changed once after threedays; water and water+dextran consumption was determined after 3 days atthe end of the dextran supplementation period.

Hemoccult (hemo FEC®, Boehringer Mannheim), presence of gross blood(blood clot around the anus) and stool consistency were determined onday 5 (and on day 6 if not positive on previous day) and on day 10.

Criteria for scoring Disease Activity Index* Occult blood Score WeightLoss (%) Stool consistency or gross bleeding 0 None Normal Negative 11-5 Loose stool Negative 2  5-10 Loose stool Hemoccult positive 3 10-15Diarrhea Hemoccult positive 4 >15 Diarrhea Gross bleeding*Disease Activity Index=(combined score of weight loss, stoolconsistency and bleeding)/3.

For hematological and microscopical tests, the animals wereanaesthetized with pentobarbital (90 mg/kg) where after the abdomen wasopened. 0.5 ml of blood was taken from the abdominal aorta and collectedin Microtainer® tubes with K₂ EDTA for hematological determination. Fordetermination of colon length, the colon was excised from colo-caecaljunction to anus, flushed with saline, placed on a non-absorbent surfaceand the colon length measured with a ruler. For histology, the distalcolon was placed in neutral buffered formaldehyde for at least 3 days.Each segment was cut into 4 transverse parts and routinely processedbefore embedding in paraffin. The Crypt scoring method [Murray et al.,1993] was as follows: grade: 0=intact crypt, 1=loss of bottom ⅓ ofcrypts, 2=loss of bottom ⅔, 3=loss of entire crypt but surfaceepithelium remains, 4=complete erosion of mucous. % area involvement:1=1-25%, 2=25-50%, 3=51-75%. 4=76-100%. The grade value score ismultiplied by the % involvement score (Maximwn score=16). The injuryscoring method (WBC in tissue) was as follows: grade: 0=none, 1=minor,2=moderate, 3=extensive. % area involvement: 1=1-25%, 2=25-50%,3=51-75%, 4=76-100%. The injury score was multiplied by the %involvement score for each of the four sections (maximum score=12).Number of lymph ‘nodes’=number of accumulations of lymph cells (persection), including normal lymph nodes: every group of lymphoid cellscontaining more than 20 cells grouped together, were considered as onesingle accumulation [Okayasu et al., 1990; Murtly et al., 1993].

FIGS. 2.7 and 2.8 show that in mice with dextran sulfate-inducedcolitis, treatment with HyPE, given orally, the parameters of diseaseactivity were considerably improved, as evidenced by overall diseasescore (FIG. 2.7) and preservation of colon length (FIG. 2.8).

These experiments demonstrate that Lipid-conjugates are effective in thetreatment of intestinal diseases and intestinal injuries.

EXAMPLE 3 Central Nervous System (CNS) Insult

The Lipid-conjugates are effective as neurotoxic agents, preventingtissue damage following physiological insult to the central nervoussystem This is demonstrated in Experiments 3.1-3.10. Ischemic stroke,trauma, infection, cancer metastases, and degenerative disease exemplifyphysiological insults in which brain tissue injury may be severe andirreversible. Tissue injury typically evokes a myriad of physiologicalresponses to stress, which in the central nervous system take the formof chemical substances released by support tissue. However, an excess ofone, or more, of these potentially neurotoxic ‘wound’ chemicals mayserve to further disrupt the healing process and contribute to the braintissue damage. Commonly accepted models for assessing theneuroprotective ability of a new drug employ preparations of brainmatrix cells (e.g., glial cells), neurotransmitter-releasing cells(e.g., PCl2 cells), and migratory blood cells (macrophages andlymphocytes) which are typically recruited to the sites of damaged braintissue. Tissue injury in the CNS is frequently compounded by localdisruption of the blood brain barrier and subsequent passage ofmigratory blood cells which may exacerbate the effects of the originalinsult and lead to extension of the tissue damage.

In response to substances associated with stress and impending injury,such as the immunogen LPS, the cytokine TNFα or the neurotoxin pardaxin,cells of the central nervous system activate a myriad of wound-responsesubstances, such as sPLA_(2,) prostaglandin (PGE₂), thromboxane (TXB₂),5-HETE, oxygen radicals, nitric oxide, or dopamine. When expressed inexcess, these substances are either themselves neurotoxic or indicativeof cotemporal neurotoxicity, thus their suppression is a frequentlychosen target for developing neuroprotective drugs.

Experiments 3.1-3.2 demonstrate Lipid-conjugate inhibition ofprostaglandin (PGE₂) release.

Experiment 3.1: Glial cell media was replaced with fresh media prior toall experiments, supplemented with 10 μg/ml LPS. Lipid-conjugates wereadded 30 minutes before exposure to LPS. The tissue cultures werefurther incubated at 37° C. for 24 h. Then the medium was collected andthe cells were incubated in fresh medium containing LPS andLipid-conjugate. After an additional 24 h, supernatants were taken fordetermination of PGE₂ content by ELISA (FIG. 3.1).

Experiment 3.2: For PC-12 cells, following incubation with the indicatedLipid-conjugate, the cells were washed then stimulated with pardaxin(PX) for 30 minutes and the amount of PGE₂ released to the medium wasdetermined by ELISA (FIG. 3.2).

Experiments 3.3 and 3.4: For demonstrating suppression of nitric oxideproduction by the lipid-conjugates, glial cell media was replaced withfresh media, supplemented with 10 μg/ml LPS. Lipid-conjugates were added30 minutes before exposure to LPS. The tissue cultures were furtherincubated at 37° C. for 2448 h. Supernatants were taken after 24 h fordetermination of NO by calorimetric measurement using the Griess reagent(FIG. 3.3). Alternately, primary mouse peritoneal macrophages weretreated with Lipid-conjugates at the indicated concentration for 30minutes (FIG. 3.4). Then LPS (1 μg/ml) was added to the culture eitherdirectly or after washing of the Lipid-conjugates. Nitric oxide wasdetermined by the Griess calorimetric method.

Experiment 3.5: For demonstration of Lipid-conjugate-induced inhibitionof soluble phospholipase A₂ (sPLA₂) release from glial cells (FIG. 3.5).Prior to all experiments, glial cell media was replaced with freshmedia, supplemented with 10 μg/ml LPS. Lipid-conjugates were added 30minutes before exposure to LPS. The tissue cultures were furtherincubated at 37° C. for 24-48 h. Culture medium samples (after 24 h)were taken for determination of PLA₂ activity by the hydrolysis ofradioactively labeled E. coli membranes. The radioactive free fatty acidreleased in this reaction was counted in a radioactivity scintillationcounter.

Experiments 3.6-3.7: To demonstrate the ability of the Lipid-conjugatesto suppress the activation of endogenous phospholipase A₂, measured asfatty acid release. Kidney pheochromocytoma (PCl2) cells weremetabolically labeled with ³H-arachidonic acid (AA) or ³H-oleic acid forat least 6 h, then washed and incubated with Lipid-conjugate asindicated for 30 minutes. The cells were then washed, stimulated withpardaxin (PX) for 30 minutes and the amount of ³H-fatty acid released tothe medium was determined in a scintillation counter (FIG. 3.6). Forrelease of oleic acid from macrophages, murine P388D₁ cells weremetabolically labeled with radiactive oleic acid, and the release ofradioactive oleic acid was determined in the presence (full circles) andabsence (empty circles) of LPS following pre-treatment with theindiucated concentration of the Lipid-conjugate, as shown in FIG. 3.7.

Experiment 3.8: To demonstrates the ability of Lipid-conjugates tosuppress dopamine (DOPA) release. PC12 cells (at confluence) were loadedwith radioactive DOPA for 4 h, then washed (in the presence ofantioxidant). The cells were then incubated with the indicatedLipid-conjugate for 30 min, then washed and stimulated with PX for 15min. The amount of labeled DOPA released to the culture medium wasdetermined in a scintillation counter (FIG. 3.8).

Experiment 3.9 For demonstrating Lipid-conjugate suppression of 5-HETErelease, PC-12 cells, under identical conditions to Experiment 3.8, areincubated with the indicated Lipid-conjugate, followed by PXstimulation. The amount of 5-HETE released was determined by ELISA (FIG.3.9).

Experiment 3.10: To demonstrate the potency of Lipid-conjugates toinhibit cell permeation through endothelial cell barrier. Using the Tcell transendothelial migration assay (FIG. 3.10) primary pig brainendothelial cells (PBEC) were plated onto collagen-coated filter,separating between upper and lower chambers. Human peripheral blood Tcells were prepared as described in Cabanas and Hogg (1993, PNAS 90:5838-5842). The T cells were maintained in recombinant human IL-2 for upto 12 days prior to use. Approximately 10⁵ T-cells were added to theupper chamber of the Transwells above the confluent PBEC monolayer andincubated at 37° C. for 5 h. Compounds for testing were also added onthe PBEC monolayer at the same time as the T cells. Electricalresistance values were measured over this period at hourly intervals. At5 hours the Transwells were briefly rinsed in warm medium and fixed inparaformaldeyde. The number of T cells which had migrated to theunderside of the filter (i.e., through the PBEC monolayer) was countedas described in the report.

These experiments demonstrate that the Lipid-conjugates are potentneuroprotective agents and useful when administered as therapy for thetreatment of brain injury in settings such as stroke, tumor, trauma,infection and degenerative disease. Additional support for the efficacyof administering Lipid-conjugates as neuroprotective agents is found inthe results of Experiment 7.4 below, demonstrating the efficacy ofadministering Lipid-conjugates for the treatment of ischemia/reperfusioninjury.

EXAMPLE 4 Multiple Sclerosis

Lipid-conjugates are effective therapy for multiple sclerosis. This isdemonstrated in experiments 4.1-4.2 below. Multiple sclerosis is adisease of white tissue in the central nervous system, marked by loss ofneurological function. The commonly accepted animal model for thisdisease is experimental allergic encephalitis (EAE) which may be inducedin rodents by subcutaneous sensitization to antigens of the nervoussystem, such as myelin basic protein. Clinical parameters are expressedby paralysis progressing from the rear limbs to the front limbs,evaluated according to the following score: Clinical signs Grade None 0Tail weakness 1 Hind limb weakness and impaired rolling 2 Hind limbparaplegia 3 Hind limb paraplegia and fore limb weakness 4 Quadriplegiaand incontinence 5 Death 6

Experiments 4.1-4.2 were perfomed to demonstrate that rats exposed toEAE-inducing agents are far less likely to develop the paralytic diseasewhen treated concurrently with Lipid-conjugate. Both experimentsemployed groups of rats in which EAE had been induced by S.C. pawinjection of 5 mg mouse spinal cord homogenate emulsified in 0.1 ml ofCFA (1:1 in PBS buffer) enriched with inactivated mycobacteriumtuberculosis 0.4 mg/ml, followed by tail vein injection of 200 ng in 0.2ml of bordetella pertussis toxin 48 hours later. In Experiment 4.1, onegroup of rats received 20 mg CMPE every other day for two weeks startingfrom the first day of the experiment. The other group received the samedose, but only from the seventh day of the experiment (after the T-cellsare activated). At the same time the respective control groups wereinjected with saline (Table 4.1). TABLE 4.1 Amelioration of EAE(Multiple Sclerosis) by CMPE Durations³ Incidence¹ Severity score²(days) EAE control 75% (6/8) 3.5 ± 2.0 3.8 ± 2.6 EAE + 20 mg/rat CMPE38% (3/8) 1.3 ± 1.7 2.1 ± 2.5 Day 1-14 EAE + 20 mg/rat CMPE 30% (3/10)1.1 ± 1.7 1.6 ± 2.5 Day 7-14

In Experiment 4.2, one group received 2 mg of CMPE every other day fromDay 1 through the 14 days of the experiment. The other group of ratsreceived 20 mg every other day from day 7 through day 14 of theexperiment (Table 4.2). TABLE 4.2 Amelioration of EAE (MultipleSclerosis) by CMPE, Low vs High Dose Duration³ Incidence¹ Severityscore² (days) EAE control 70% (7/10) 2.9 ± 1.4 3.7 ± 1.0 EAE + 2 mg/ratCMPE, 50% (5/10) 1.1 ± 0.5 4.4 ± 0.8 Day 7-14 EAE + 20 mg/rat CMPE, 20%(2/10) 0.5 ± 1.1 2.7 ± 1.4 Day 7-14

Both experiments show that therapy with Lipid-conjugates results in aless severe course of disease and more complete recovery of motorfunction, as judged by the percentage of rats showing paralysis(incidence¹), the degree of paralysis and progression towards the frontlimbs (severity score²), and the duration of paralysis until recovery(duration³). In addition, the results presented in Table 4.2 demonstratethat the therapeutic effect of the Lipid-conjugates is dose-dependent.

Additional support for the efficacy of Lipid-conjugates in multiplesclerosis may be found in Experiments 3.1, 3.3-3.5 and 3.10, above,wherein the neuroprotective effect of the Lipid-conjugates isdemonstrated.

EXAMPLE 5 Skin Diseases, Contact Dermatitis and Psoriasis

The Lipid-conjugates are effective in the treatment of cutaneoushypersensitivity reactions and psoriasis. This is demonstrated inExperiments 5.1-5.5. Skin hypersensitivity reactions may occur inresponse to virtually any material and may present in both acute andchronic forms. Systemic sensitization to an antigen followed by itslocal application is a widely-accepted system for invoking the delayedtype hypersensitivity response attributed to the mechanism of contactdermatitis. Psoriasis is a common form of dermatitis marked byplaque-like formations, evident on extensor surfaces and, as ahyperproliferative disorder of epithelial cells, drug therapies atetypically examined in cell cultures obtained from sufferers of thecondition.

Experiments 5.1-5.4 demonstrate that treatment of the animals afflictedwith a hypersensitivity reaction readily respond to the administrationof Lipid-conjugates, whether applied intraperitoneally (Table 5.1),subcutaneously (Table 5.2), or topically (Tables 5.3-5.4), as bothprophylactic and acute therapy.

Three modes of administration were performed: 1) The Lipid-conjugate insaline was injected intraperitoneally daily beginning day 0 until day 6(Table 5.1); 2) The Lipid-conjugate in saline was injectedsubcutaneously into the ear (adjacent to the challenged area) in twoinjections, either 3 h before application of oxalozone to the ear or 1 hafter application of oxalozone to the ear (Table 5.2); 3) EtOH:H₂O 1:1was applied topically to both ears on top of the challenged area dailybeginning day 0 until day 6 (Table 5.3); 4) the Lipid-conjugate wasapplied topically only to the right ear for 5 times 4-6 hours followingthe challenge (Table 5.4) using either 20 μL of 0.1% DEXPE in 50% EtOHor 20 μl of Dermovat (steroid ointment). In all experiments controlGroup A (late sensitized only) was treated by topical application ofoxalozone to both sides of the ear 24 hours before measuring itsswelling. Group B (fully sensitized+saline or EtOH 50% was treated bytopical application of oxalozone to the shaved stomach and then on day 6by topical application of oxalozone to both sides of the ear. Swellingwas measured in 0.1 mm by subtracting normal ear width of eachindividual mouse from the width after treatment. Percent inhibition wascalculated by the net swelling of the Lipid-conjugate-treated ear (overthat of the control group A), divided by the net swelling of thefully-sensitized ear. As shown in Tables 5.1-5.4, in all cases,treatment with the Lipid-conjugates clearly reduced ear swelling inDTH-induced mice. Of particular interest are the results presented inTable 5.4, showing that although the topical administration of the drugwas unilateral in both cases, the steroid affected both ears, while thetopically applied Lipid-conjugate affected only the area to which it wasapplied, indicative of a lack of systemic infiltration of theLipid-conjugate in this context. TABLE 5.1 Attenuation of Dermal DTHResponse by CMPE - Intraperitoneal Administration Swelling aftersensitization − Swelling of normal ear (0.1 mm) No. of Mean ± S.D.Percent Group Treatment Mice (n = 12) inhibition A Control (latesensitized) 6 1.8 ± 1.0 — B Fully sensitized + saline 6 18.5 ± 0.97 — CFully sensitized + CMC 6 19.8 ± 1.13 — 40 mg (0.4 μmol/kg) D Fullysensitized + CMPE 6 7.9 ± 1.37 66 40 mg (0.4 μmol/kg) E Fullysensitized + 6 6.5 ± 1.35 74 betamethasone 5 mg(15 μmol/kg)

TABLE 5.2 Attenuation of Dermal DTH Response by CMPE - SubcutaneousAdministration Swelling after sensitization − Swelling of normal ear(0.1 mm) No. of Mean ± S.D. Percent Group Treatment Mice (n = 12)inhibition A Control (late sensitized) 5  4.1 ± 0.82 — B Fullysensitized + saline 5 18.3 ± 0.82 — C Fully sensitized + CMC 5 13.5 ±2.17 35 (carrier polymer only) 40 mg (0.4 μmol/kg) D Fully sensitized +CMPE 5  5.9 ± 1.52 87 40 mg (0.4 μmol/kg) E Fully sensitized + 5  8.1 ±1.19 72 betamethasone 1 mg (3 μmol/kg)

TABLE 5.3 Attenuation of Dermal DTH Response by DEXPE - TopicalAdministration Swelling after sensitization − Swelling of normal ear(0.1 mm) No. of Mean ± S.D. Percent Group Treatment Mice (n = 12)inhibition A Control 5  1.5 ± 0.70 — (late sensitized only) B Fullysensitized + saline 5 24.3 ± 1.56 — C Fully sensitized + 5 24.4 ± 2.4  —Dextran (carrer polymer only) (0.5 μmol/kg) D Fully sensitized + 5 12.17± 1.52  53 DEXPE (0.5 μmol/kg) E Fully sensitized + 5 10.6 ± 0.84 60betamethasone (3 μmol/kg)

TABLE 5.4 Attenuation of Dermal DTH Response by DEXPE - UnilateralTopical Administration vs Steroid Preparation Swelling aftersensitization − Swelling of normal ear Percent No. of (0.1 mm) Mean ±S.D. (n = 10) inhibition Group Treatment mice Left ear Both ears Rightear Left ear Right ear A Control, 10  1.0 ± 2.0 — — (late sensitizedonly) B Fully sensitized + vehicle 10 23.0 ± 4.0 — — (dextran) C Fullysensitized + DEXPE 7 20.0 ± 1.0 11.0 ± 1.0 14 46 (0.5 μmol/kg), on rightear only. D Fully sensitized + betamethasone 7  7.0 ± 1.0  7.0 ± 1.0 6363 (3 μmol/kg, dermovat) on right ear only.

Experiment 5.5: To show that Lipid-conjugates effectively inhibit theproliferation of cultured psoriatic skin fibroblasts and Swiss 3T3cells. Fibroblasts of human psoriatic skin (dermis) cells, (fullcircles) or Swiss 3T3 cells (empty circles) were treated with CMPE atthe indicated concentration for three days, after which the cells werecounted (FIG. 5.1). The cell number of the control, untreated group atthe end of the three day incubation was taken as 100%. For comparison,carboxymethylcellulose was tested alone (square).

These experiments demonstrate that Lipid-conjugates are effectiveremedies for the management of various forms of dermititis includingskin hypersensitivity reactions and psoriasis. Additional support forthe applicability of the Lipid-conjugates the treatment of skindiseases, is provided by Examples 9, 11 and 12, showing that theLipid-conjugates protect from oxidants and suppress the production ofcytokines and lipid mediators, which are involved in the pathogenesis ofskin injuries.

EXAMPLE 6 Cardiovascular Disease

The Lipid-conjugates are effective therapy for ischemic vasculardisease, atherosclerosis, and reperfusion injury. This is demonstratedin Experiments 6.1-6.3.

A prominent feature in the pathogenesis of atherosclerosis is theaccumulation of blood lipoproteins, such as oxidized LDL (oLDL), incells lining vascular walls, and the proliferation of cells lining andwithin vascular walls, such as smooth muscle cells. The resultantnarrowing of the blood vessel lumen at the site of the atheroscleroticlesion may give rise to varying degrees of tissue ischemia. Whileischemic events may be reversible, either spontaneously or throughmedical intervention, the process of tissue injury may persist to thestage of reperfusion injury, in which the previously ischemic tissue isstill at risk for damage, through several mechanisms, includingoxidative damage.

Experiment 6.1: LDL-PLA₂. Endogenous LDL-phospholipase A₂ (PLA₂)hydrolyzes LDL-phospholipids to form lyso-phospholipids, which arechemotactic and facilitate LDL oxidation and uptake by blood vessel wallcells. For demonstrating that the Lipid-conjugates inhibitLDL-associated PLA₂ activity, LDL (0.1 μM) was incubated for 15 min at37° C. in the absence or presence of HYPE, HEPPE or CMPE at theconcentrations indicated (FIG. 6.1). At time zero C₆-NBD-PC (0.5 μM) wasadded to the dispersion. This resulted in an instantaneous increase offluorescence intensity (due to incorporation of NBD into lipidic cores).When LDL was incubated alone the increase of fluorescence was followedby time-dependent decrease of fluorescence intensity that can beattributed to hydrolysis of the LDL-associated PLA (and subsequentdeparture of the resultant NBD-caproic acid from the LDL particle to theaqueous medium). When LDL was incubated in the presence of HYPE, HEPPEor CMPE this time-dependent decrease was fully or partially inhibited.

Experiments 6.2-6.3: To demonstrate that the Lipid-conjugates inhibitLDL uptake by cultured macrophages and in whole animals, human LDL(isolated by the conventional method of floatation) were subjected toCu²⁺-induced oxidation, and labeled with ¹²⁵I. Confluent J774macrophages were incubated with 100 μM ¹²⁵I-oLDL and Lipid-conjugate atthe indicated concentration in PBS buffer (pH=7.4) supplemented with0.5% BSA, for 3 h. The cells were then washed 4 times with the PBS/BSA,and subjected to lysis by 0.1 N NaOH for 30 min. The cell lysate wascollected and the 125I content was determined in a radioactivity counter(Table 6.1). TABLE 6.1 Inhibition of Oxidized LDL Uptake in macrophagesby HYPE and HEPPE Cell-associated Treatment ¹²⁵I-oLDL (DPM × 10⁻³) %Inhibition Control 92.2 ± 4.0 10 μM HYPE 20.9 ± 1.7 78% 20 μM HEPPE 59.2± 8.3 37%

Experiment 6.3: Uptake of oLDL in-vivo: Rats weighing 200 g wereinjected I.V. with 0.4 ml saline containing 250 nmole of Cu²⁺-inducedoxidized LDL labeled with ¹²⁵I, and 200 mmole of HYPE. Blood sampleswere drawn at the indicated time intervals and the ¹²⁵I radioactivity inthe plasma was counted (FIG. 6.2).

These experiments demonstrate that administration of Lipid-conjugates iseffective therapy in the treatment of cardiovascular disease, includingatherosclerosis. Additional support for the capacity of theLipid-conjugates to treat cardiovascular diseases is provided inExperiments 7.1-7.3 and Experiments 9.3 below, showing that theLipid-conjugates inhibit proliferation of smooth muscle cells, andprotect LDL from oxidative damage.

EXAMPLE 7 Prophylaxis for Invasive Surgical Procedures, IncludingCatheterization

The Lipid-conjugates are effective in the treatment and prophylaxis forcardiovascular disease in many settings, including atherosclerosis, asdescribed above, as well as in the setting of stenosis and restenosisinduced by ischemia/reperfusion injury. The lipid-conjugates areeffective in preventing the formation of stenotic lesions as may occurin the course of invasive surgical procedures which involve manipulationof vascular organs, in particular vascular catheterization.

Since the proliferation of vascular smooth muscle cells (SMC) is theprocess leading to blood vessel stenosis, the Lipid-conjugates wereassessed for their effect on this process.

Experiments 7.1-7.3 demonstrate the anti-proliferative effects of theLipid-conjugates on bovine aortic smooth muscle cells, unstimulated orstimulated by thrombin, and on the proliferation of human venous smoothmuscle cells.

Experiment 7.1: For unstimulated cells, bovine aortic smooth musclecells were seeded at 7×10³ cells per well (in 24-well plates), in DMEMsupplemented with 10% FCS, in the absence or presence of HYPE-40 orHYPE-80 (enriched with PE), grown for 72 h, and counted in Coulter (FIG.7.1).

Experiment 7.2: For stimulated cells, bovine aortic smooth muscle cellswere grown under the conditions as above for 48 h, followingpre-incubation for 6 h, as indicated, with either thrombin, fetal calfserum, Lipid-conjugate, or both. Cell growth is represented as theamount of thymidine incorporation (FIG. 7.2).

Experiment 7.3: SMC from human saphenous vein, were inoculated at8×10⁴/cells/5 mm culture dish, in DMEM supplemented with 5% fetal calfserum and 5% human serum. A day later the cells were washed andincubated in the same culture medium in the absence (control) orpresence of the Lipid-conjugate (HEPPE) or its polymeric carrier(heparin, at the same concentration as the HEPPE). After 5 days thecells were harvested (by trypsinization) and counted (FIG. 7.3). Eachdatum is mean±SEM for 3 replications (the same results were obtained ina second reproducible experiment). *p<0.005.

Experiment 7.4: Ischemia/reperfusion injury: As noted above, the injuryinduced by ischemia and reperfusion, is the major stimulant for stenosissubsequent to catheterization, surgery or other procedures that involvevascular obstruction and occlusion. To demonstrate the ability of theLipid-conjugates to ameliorate this injury, they were tested forinhibition of white cell adhesion and extravasaion, which expressischemia/reperfusion injury to blood vessels. Leukocytes were labeled invivo by I.V. injection of rhodamine. Ischemia was applied to exposedcremaster muscle in rats (in situ) for 90 min, then blood flow wasrestored for reperfusion. The fluorescent-labeled leukocytes adherent toblood vessel walls (FIG. 7.4A) and those extravasated to theextravascular space (FIG. 7.4B) were videotaped and counted at theindicated time point during the reperfusion period. Lipid-conjugates (10mg. 100 g body weight) were injected IV. 40 min and 10 min prior toinduction of ischemia. FIG. 7.4A and 7.4B show that administration ofLipid-conjugates efficiently suppresses the ischemia/reperfusion-inducedadhesion and extravasation of leukocytes. Each datum is mean±SEMobtained from 5 rats with HYPE and 3 rats with HEPPE. p<0.005.

Experiment 7.5: Another expression of damage to blood vessel wallendothelium is adhesion of red blood cells (RBC) to endothelial cellsupon their activation by oxygen radicals, lipid mediators or cytokines(produced subsequent to ischemia reperfusion injury). RBC adherencefurther facilitates vascular occlusion. For demonstrating the protectiveeffect of Lipid-conjugates on endothelium, bovine aortic endothelialcells were exposed to either tumor necrosis factor (TNF-α),phospholipase A₂, arachidonic acid, or hydrogen peroxide, and thenassayed for cytodamage, as judged by adhesion of red blood cells as anindex of endothelial intactness. Bovine aortic endothelial cells (BAEC)were pre-incubated for 30 min with either 5 μM CMPE or 20 μM DEXPE, thenwashed and stimulated for 18 h with TNF, ArAr, or PLA₂ at the indicatedconcentration. For stimulation with H₂O₂, the cells were treated withH₂O₂ for 20 min, then washed and incubated in the control culture mediumfor 18 h. The BAEC were washed and incubated with human red blood cells(RBC) for 30 min. The cultures were washed and the RBC which remainedadhering to the BAEC were counted under a microscope (FIG. 7.5).

Experiment 7.6: Balloon-induced stenosis in rats: To demonstrate theefficacy of Lipid-conjugates in protocols for balloon-induced stenosisin rats, in the carotid artery by both systemic (Table 7.1) andintravenous infusion administration. Rats were pre-treated with I.P.injection of 10 mg/100 g body weight of HYPE in PBS, or PBS alone, 1day, and also 1-2 hours before injury. Injury was achieved using thestandard Fogarty catheter. The rats were injected with the same amountof drug or vehicle every day for 3 days, and then every other day, for atotal of 8 injections. Rat were sacrificed on the 14^(th) day, thearteries were processed according to standard procedure. Half of therats were injected with bromodeoxyuridine (BrdU), fixed with formalinand triton, and processed for BrdU staining, and areas of the indicatedvascular structures measured for comparison (Table 7.1). The distal leftcommon and external carotid arteries were exposed through a midlineincision in the neck. The left common carotid artery was denuded ofendothelium by the intraluminal passage of a 2F Fogarty balloon catheter(Baxter, Santa Anna, Calif.) introduced through the external carotidartery. The catheter was passed three times with the balloon distendedsufficiently with saline to generate a slight resistance. The catheterwas then removed and a polyethylene tube (PE-10) connected to a syringewas introduced into the common carotid artery. A segment of the commoncarotid artery was temporarily isolated by sliding ligature and vascularclamp. Approximately 50 μl of solution containing 10 mmole of CMPE wasinjected into isolated arterial segment and left in place for 15 min.The drug solution was then evacuated and the external carotid artery wasligated. The rats were sacrificed 2 weeks later, and the percent ofluminal stenosis (in the damaged area) was determined by histologicalmeasurement of neointima (N) to media (M) area ratio (Table 7.1). TABLE7.1 Inhibition of Balloon-Induced Stenosis in Rats by Lipid-Conjugates %stenosis Experiment Treatment (Mean ± SEM) P N/M P I.P administrationUntreated (n = 7) 53.96 ± 4.11 0.003 1.64 ± 0.12 0.001 HyPE (n = 6)53.96 ± 2.89  1.0 ± 0.08 I.P. administration Untreated (n = 6) 41.53 ±4.84 0.023 1.16 ± 0.12 0.036 CMPE (n = 8) 21.89 ± 5.42 0.64 ± 0.17Intra-arterial Untreated (n = 4) 53.12 ± 12.8 0.052 1.61 ± 0.17 0.008Administration CMPE (n = 6) 29.64 ± 2.17 0.99 ± 0.08

These experiments demonstrate that administration of Lipid-conjugatesare effective therapy in the treatment of cardiovascular disease, by aplurality of mechanisms, including inhibition of vascular smooth musclecell proliferation, uptake of lipoprotein, oxidative stress, andleukocyte activation in models of ischemia and reperfusion.Administration of Lipid-conjugates is of both prophylactic and acutetherapeutic benefit when administered in the course of invasive arterialprocedures, particularly balloon angioplasty.

EXAMPLE 8 Invasive Cellular Proliferative Disorders

The Lipid-conjugates are effective therapy for cellular proliferativedisorders, such as cancer. This is demonstrated in experiments 7.1-7.3above and 8.1-8.8 below. The process of cancer spread entails multipleevents, each of these is a worthy target for inhibitory drug action,including the rate of cell-proliferation, the rate of spread throughblood vessels, the rate of invasiveness through contiguous andnon-contiguous (metastases) tissues, and the rate of production of newblood vessels to supply the cancerous growth. Cancer cells frequentlyproduce intracellular matrix tissue degrading enzymes which serve toenhance their invasive potential Cancer is thus a multiphasic diseaseinvolving the process of tissue invasiveness, spread through tissuechannels, angiogenesis and tumor vascularization. These latter processesdepend upon the rates of proliferation of endothelial cells and smoothmuscle cells.

Experiment 8.1-8.3 demonstrate that the Lipid-conjugates inhibit theproduction and activities of enzyme that break the basal membrane andenable the invasion of cancer cells, such as collagenase(metaloproteinase=MMP), heparinase and hyaluronidase:

Experiment 8.1: To demonstrate the Lipid-conjugate effect oncollagenase, HT-1080 (fibrosarcoma) cells were incubated for 24 h withHYPE at the indicated concentration. The culture medium was thencollected and its collagenase activity was determined by a zymographicassay. Each datum is average of two plates (FIG. 8.1).

Experiment 8.2: To demonstrate the ability of the Lipid-conjugates toinhibit hyaluronidase activity, hyaluronic acid (HA) in PBS (0.75 mg/ml)was interacted with hyaluronidase (15 U/ml) in the absence or presenceof HYPE, at the indicated concentration for 1 h. HA degradation wasdeterinined by the change in the viscosity of its solution (FIG. 8-2).

Experiment 8.3: To demonstrate the inhibition of heparinase activity byLipid-conjugates, BGM cells were incubated overnight with 50 μCi ³⁵SO₄²− per well (to label the cell surface glycosaminoglycans). The cellsthen were washed 3 times with PBS before treating with 5 units ofheparinase I in 200 μl PBS for 3 h. The medium was collected and its ³⁵Scontent was counted (FIG. 8.3).

Experiment 8.4: For showing the ability of the Lipid-conjugates toinhibit the invasion of tumor cells through basement membrane, thechemoattractant invasion assay was used: Polycarbonate fibers, 8 μm poresize, were coated with 25 μg of a mixture of basement membranecomponents (Matrigel) and placed in modified Boyden chambers. The cells(2×10⁵) were released from their culture dishes by a short exposure toEDTA (1 mM), centrifuged, re-suspended in 0.1% BSA/DMEM, and placed inthe upper compartment of the Boyden chamber. Fibroblast conditionedmedium was placed in the lower compartment as a source ofchemoattractants. After incubation for 6 h at 37 C, the cells on thelower surface of the filter were stained with Diff-Quick (AmericanScientific Products) and were quantitated with an image analyzer(Optomax V) attached to an Olympus CK2 microscope. The data areexpressed relative to the area occupied by untreated cells on the lowersurface of the filter. (Albini et al., A Rapid In Vitro Assay forQuantitating the Invasive Potential of Tumor Cells. Cancer Res.47:3239-3245, 1987). FIG. 8.4 demonstrates the Lipid-conjugate abilityto attenuate cancer cell invasiveness.

Experiment 8.5: For demonstrating Lipid-conjugate effect onproliferation of endothelial cells, bovine aortic endothelial cells wereplated in culture dishes for 6 h, then washed to remove unattachedcells. The remaining attached cells were incubated in the absence(control) or presence of Lipid-conjugates at the indicatedconcentration, and stimulated with VEGF (vascular endothelial growthfactor) for 48 h. The cells were then washed, collected bytrypsinization and counted in a Coulter counter. The results aremean±S.D. for 3 replications. *p<0.005 (FIG. 8.5).

Experiment 8.6: Similar effect was observed with human bone marrowmicrovascular endothelial cells (UBMEC), stimulated with differentgrowth factors, namely VEGF, b-FGF (fibroblast growth factor), or OSM(oncostatin), as shown in FIG. 8.6.

Experiment 8.7: The capacity of the lipid-conjugates to controlangiogenesis is illustrated in FIG. 8.7. This Figure demonstrates theinhibitory effect induced by HyPE on capillary tube formation by HNMEC,in a three-dimensional fibrin gel, stimulated by the above growthfactors. HyPE (20 μM) or hyaluronic acid (the carrier without the lipidmoiety) were added to the HBMEC-coated beads in the fibrinsimultaneously with the growth factors. Line A: b-FGF (25 ng/ml), lineB: VEGF (20 ng/ml) and line C: OSM (2.5 ng/ml). Column A: without HyPE,Column B: with HyPE (20 μM).

The corresponding quantitation of the capillary formation is presentedin Table 8.1: TABLE 8.1 HyPE inhibits bFGF-, VEGF- and OSM-stimulatedCapillary Tube Formation in a three-dimensional fibrin Gel. Length (μm)Width (μm) Treatment −HyPE +HyPE −HyPE +HyPE Control 232.23 ± 56.13 80.31 ± 30.59***  9.42 ± 1.65  8.32 ± 1.47 BFGF 533.92 ± 65.02 266.73 ±23.17*** 15.83 ± 2.96 11.21 ± 1.52* VEGF 511.09 ± 72.05 215.68 ±31.22*** 14.86 ± 1.46  9.32 ± 1.18** OSM 518.82 ± 58.49 234.85 ±36.32*** 16.89 ± 1.89 10.02 ± 1.00***Each datum is mean ± SEM of 3 experiments;5 beads were examined in each field.***p < 0.005,**p < 0.01,*p < 0.05

Experiment 8.8: Effect of Lipid-conjugates on mouse lung metastasesformation induced by mouse melanoma cells: 10⁵ B16 F10 mouse melanomacells were injected I.V. into a mouse (20-25 g). Three weeks later thelungs were collected and the metastases on the lung surface counted. TheLipid-conjugate effect, illustrated in FIG. 8.8, was examined asfollows: In experiment 1, the indicated Lipid-conjugates was injectedI.P. (1 mg/mouse) 5 times a week for 3 weeks starting on day 1 (total of15 injections) (FIG. 8.8-I). In FIG. 8.8-II, HYPE (selected subsequentlyto experiment 1) was injected I.P. (1 mg/mouse) as follows: A. 5 times aweek for 3 weeks starting on day 1 (total of 15 injections); B. 5 timesa week for 2 weeks starting from week 2 (total of 10 injections); C. Oneinjection (I.P.) simultaneously with I.V. injection of the melanomacells. D=Mice injected (I.P.) with hyaluronic acid alone (without PE), 5times a week for 3 weeks, starting on day 1 (total of 15 injections).Each group included 6 mice. *p<0.0001, **p<1.10-5, ***p<2.10-7.

In addition, Experiments 7.1-7.3 above also demonstrate the capacity ofthe Lipid-conjugates to control the proliferation of smooth musclecells, which is essential for tumor vascularization subsequent tocapillary formation by endothelial cells.

Taken together, the experiments described above, demonstrate thatadministration of the Lipid-conjugates are effective therapy in thetreatment of cancer growth and metastasis, by a plurality of mechanisms,including suppression of cell proliferation, invasion of cancer cells,angiogenesis and metastasis formation and tumor vascularization.

EXAMPLE 9 Anti-Oxidant Therapy

The Lipid-conjugates are effective therapy for preventing oxidativedamage. This is demonstrated in Experiments 9.1-9.3. The noxious effectof peroxide free radicals on living tissue is known as oxidative damage.When cell membranes are the targets for this damaging process, membranedysfunction and instability result. Oxidative damage to blood proteins,particularly blood lipid proteins, results in their over-accumulation incells lining the vasculature, thus contributing to atherogenesis. Infact, oxidative cell damage is a major mechanism attributed to theprocess of aging or senescence.

Oxidative damage to proteins or cell membranes is commonly assessed byexposing these tissues to hydrogen peroxide produced by the enzymeglucose oxidase (GO), in the absence or presence of additional membranedestabilizing agents, such as PLA₂, or by exposure to divalent cations,such as copper.

Experiments 9.1-9.3 demonstrate the ability of Lipid-conjugates topreserve cells from oxidative damage, as judged by the cells' retentionof both arachidonic acid and of low molecular weight intracellularsubstances.

Experiment 9.1: Confluent BGM (green monkey kidney epithelial cells)were labeled with ³H-arachidonic acid. The cells were treated with CMPEfor 30 min prior to treatment with GO and PLA) (0.5 u/ml) (FIG. 9.1).

Experiment 9.2: BGM cells were labeled with ³⁵SO₄ overnight. The cellswere washed with DMEM (containing 10 mg/ml BSA) 4 times with PBS. Thecells were then incubated in DMEM supplemented with GO (an H₂O₂generation) for 90, and the culture medium was collected and counted for³⁵S radioactivity. For treatment with CMPE cells were incubated withCMPE, at the indicated concentration for 30 min prior to introduction ofGO. Each datum is MEAN+SEM for 5 replications. *p<0.005; **p<0.001 (FIG.9.2).

Experiment 9.3: For demonstrating the ability of Lipid-conjugates toinhibit the oxidation of blood lipoprotein. LDL (0.1 μM) was incubatedin the absence and presence of various concentrations of HYPE or HA at37° C. At time zero 5 μM CuCl₂ was added to the dispersions and themixtures were continuously monitored for oxidation products at 245 nm(FIG. 9.3). The absorbance at 245 (OD units) is depicted as a functionof time (Shnitzer et al., Free Radical Biol Med 24; 1294-1303, 1998).

Additional support for the anti-oxidant capacity of the Lipid-conjugatesis provided by Experiment 7.4 above, showing their inhibitory effect onischemia/reperfusion-induced activation of white cells.

These experiments demonstrate that administration of Lipid-conjugates iseffective therapy in the prevention of tissue damage induced byoxidative stress (associated with free radical and hydrogen peroxideproduction) by a plurality of mechanisms, including inhibiting theoxidation of lipoprotein, as well as their uptake (Experiment 6.3),inhibiting arachidonic acid release, and preserving the integrity ofcell membranes (inhibiting GAG degradation), including red blood cellmembranes, as described below.

EXAMPLE 10 Hemolysis

The Lipid-conjugates are effective therapy in the treatment andprevention of hemolysis. This is demonstrated in Experiments 10.1.Hemolysis, the breakdown of red blood cells (RBC), may be either aprimary disease in itself, or a syndrome associated with another diseaseor physiological insult. A commonly accepted model for assessing themembrane-stabilizing effect of a drug is to incubate red blood cells inthe presence of known membrane destabilizing agents and to detect forthe release of hemoglobulin into the extracellular medium.

Experiment 10.1: To demonstrate that the Lipid-conjugates serve tomaintain the stability of human red blood cells exposed tomembrane-destroying agents. Human RBC were washed in saline andsuspended in Hanks buffer (pH-7.4). Hemolysis was induced in the absenceor presence of Lipid-conjugates (10 μM), as indicated, by treatment witheither streptolysin O (SLO) 5 U/ml, streptolysin S (SLS) 25 U/ml, orlysophosphatidylcholine (lyso-PC) 5 μg/ml for 20 min. The cell membraneswere spun and the hemoglobin content in the supernatant was determinedby measuring the O.D. at 540 nm (Table 10.1). TABLE 10.1 Prevention ofHemolysis by HYPE, CMPE and HEPPE HEMOLYSIS (O.D. AT 540 nm)Lipid-conjugate SLO SLS Lyso-PC None 1.000 1.000 1.000 HA 1.000 1.0001.875 HYPE-30* 0.650 0.750 0.335 HYPE-60 0.012 0.005 0.017 HYPE-1100.005 0.002 0.012 CMPE-60 0.012 0.005 0.002 CMPE-110 0.002 0.002 HEPPE0.002 1.100 0.002*The number expresses the amount of nmoles lipid conjugated to 1 mg ofpolymer.

These experiments demonstrate that the Lipid-conjugates are effectivetherapy in the treatment of hemolysis and of value as preservatives inblood product storage. Thus Lipid-conjugates are demonstrated to haveutility in maintaining hematocrit and in blood-banking.

EXAMPLE 11 Sepsis

The Lipid-conjugates are effective therapy in the treatment ofbacteremia-induced shock, otherwise known as septic shock, sepsis orsepticemia. This is demonstrated in Experiments 11.1-11.8.

Sepsis is characterized by enhanced levels of cytokines such as Tumornecrosis factor (TNFα) and interleukine-1 (IL-1), IL-6 and IL-8, andendothelial cell adhesion molecules, such as ICAM-1 and E-Selectin Theseare involved in the pathogenesis of septic shock, being released bothlocally and systemically to produce noxious and irreversible effects ontissue integrity and systemic hemodynamics. Exposure of cells to thebacterial lipopolysaccharide (LPS) and Lipoteichoic acid (LTA)immunogens comprises a commonly-used model system for assaying theresponse of these agents to septicemic conditions.

Experiment 11.1: To demonstrate the ability of the lipid-conjugates toinhibit elaboration of TNF-α in human tissue, fresh heparinized (12.5U/ml) human venous blood from healthy blood donors was diluted 1:3 withmedium RPMI-1640, supplemented with 200 mM glutamine, 200 U/mlpenicillin and 200 U/ml streptomycin. Fractions (300 μl) of 1:3 dilutedblood were distributed in 24 well Multidisk plates (Nunclon). Bloodsamples were pre-incubated (30 min at 37° C.) in a humidified atmosphereof 6% CO₂ with 100 μl of compound or solvent before being stimulated bythe addition of 100 μl of lipopolysaccharide E. coli 026:B6 (LPS) at afinal concentration of 100 ng/ml. After 6 h incubation, the 24 wellplates were spun down (2000 rpm×10) and assayed for cytokine content byELISA. The various HyPEs differed in their phosphate content (FIGS.11.1-II and 11.1-II).

Experiment 11.2: Sepsis in-vivo: To demonstrate the lipid-conjugatecapacity to ameliorate sepsis, they were tested for their effect onendotoxin-induced sepsis in a rat model. To this end the followingprocedures were performed:

Since endotoxins, administered to animals, produce cardiovascular andmultiorgan disorders that are similar to clinical sepsis, in the presentstudy a rat-model was developed to test possible Lipid-conjugateseffects on mediator production and mortality in endotoxin-inducedSepsis. Rats were intraperitoneally (I.P.) or intravenously (I.V.)injected with the Lipid conjugates (specifically HyPE, 100 mg/kg)dissolved in sterile saline or with sterile saline alone as placebo. 3hours thereafter all rats received LPS (15 mg/kg) i.p. (Escherichia coli111:B44, Sigma, Deisenhofen, Germany). In rats that were pretreated withHyPE, LPS was injected together with a refreshing dose of HyPE (50mg/kg). The concentration of HyPE was determined by extrapolation fromthe previous in-vitro and in-vivo studies (cited above). The effect ofHyPE on LPS injected rats was observed over a time period of 48 hours.As show in in FIG. 11.2, treatment with the Lipid conjugate HyPEmarkedly reduced the mortality rate among septic rats.

Experimnet 11.3: For determination of serum levels of TNF-α and IL-6,rats were either pretreated for defined time periods with a priming doseof Lipid conjugates (HyPE or CSAPE as described above), or untreated.Thereafter, the animals received LPS (7.5 mg/kg) i.p. or LPS+LTA i.p.(5+5 mg/kg) (Staph. aureus, Sigma, Genmany) alone or together with HyPE(50 mg/kg) or CSAPE (50 mg/kg). Rats that were treated with neitherLipid-conjugates nor with LPS were used as negative control. In otherexperiments HyPE was intravenously (i.v.) administered (100 mg/kg)simultaneously with an i.p. injection of LPS. Blood samples werecollected 60 min, 6 hours and 24 hours after LPS-injection to assesscytokine concentrations. All cytokines were measured in separated serumby ELISA Immunoassays (R&D Systems GmbH, Wiesbaden, Germany) accordingto the instructions of the manufacturer. FIG. 11.3 demonstrtaes thatcytokine level in the serum of septic rats is markedly reduced bytreatment with Lipid-conjugates.

In Experinment 11.4 HyPE was given inravenously (I.V.) at the same timewhen LPS was given I.P. (while in Experiment 11.3 HyPE was given I.P. 3h prior to LPS). As shown in FIG. 11.4, endotoxin-induced cytokineproduction was suppressed as well by this mode of treatment as with theLipid-conjugate.

In Experiment 11.5, sepsis was induced by LPS (gram-positive endotoxin,5 mg/kg) and lipoteichoic acid (LTA, gram-negative endotoxin, 5 mg/kg).FIG. 11.5 demonstrates that the Lipid-conjugates are effective also insuppression of cytokine production induced by this combination ofendotoxins.

Experiment 11.6: Lipid-conjugates inhibit endotoxin-induced cytokinemRNA expression. For RNase protection assay (RPA): Rat lung and kidneywere removed from Lipid-conjugate-treated or untreated rats 24 hoursafter Sepsis induction for total RNA isolation using Trizol reagent(Gibco BRL, Eggenstein, Germany). The concentration of RNA in eachsample was assessed spectrophotometrically. To evaluate specific RNAlevels in rat lung and kidney, a multiprobe RPA-kit was used (riboQuant,PharMingen, Heidelberg, Germany) according to manufacturer'sinstructions. Briefly, a set of ³²P-labeled RNA probes synthesized fromDNA templates using T7 polymerase was hybridized with 7 μg of total RNA,after which free probes and single-stranded RNA were digested withRNase. Undigested probes and digested samples were loaded on to a 5%denaturing polyacrylamide gel, dried and exposed to a Kodak X-apartfilm. The expression of each specific mRNA was related to twohousekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) andL32, to exclude differences in the amount of RNA that was hybridized.The following templates for rat cytokines were used in the presentstudy: IL-1-α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-α, TNF-β,IFN-γ, L32 and GAPDH. As shown in FIG. 11.6, treatment withLipid-conjugate inhibited the endotoxin-induced cytokine geneexpression, respective with FIG. 11.3.

Experiment 11.7: RNA expression of iNOS and secretory PLA₂ Type II(sPLA₂II): For Polymerase Chain Reaction, Total RNA, isolated from ratlung and kidney, was subjected to DNAse digestion (Gibco BRL,Eggenstein, Germany) to remove possible contaminations of genomic DNA. 1μg of total RNA was reverse-transcribed to cDNA using SuperScript™ IIPreamplification System (Gibco BRL, Eggenstein, Germany), essentially asrecommended by the manufacturer's instructions. Amplification of 0.5 μlcDNA was performed in a total volume of 25 μl containing 19.6 pmol ofeach primer (Table 1), 5 mM dNTPs, 2.5 U Taq Polymerase, 10 mM Tris HCl,7.5 mM KCl, 1.5 mM MgCl₂. PCR reactions were initiated at 94° C. for 3min, followed by varying cycles of amplification, each consisting ofdenaturation at 94° C. for 1 min, annealing at 60° C. for iNOS and 65°C. for sPLA₂-IIA and primer extension 72° C. for 2 min. At the end ofthe amplification cycles the products were incubated for 10 min at 72°C. In each experiment for each PCR reaction two controls were included,i.e., omitting reversed-Transcriptase from the cDNA synthesis reactionor omitting cDNA from the amplification reaction. PCR products wereseparated on a 1% agarose gel. FIG. 11.7 demonstrates that the Lipidconjugate ability to suppress the endotoxin-induced gene expression ofsPLA₂ IIA and iNOS.

Experiment 11.8: Inhibition of adhesion molecule expression: Fordetermination of ICAM-1 expression in rat tissues(Immunohistochemistry), Cryostat sections of pulmonal and renal tissuewere analyzed by an indirect immunoperoxidase technique. Briefly,ethanol-fixed sections were incubated with primary antibody againstICAM-1 for 1 hour, washed and incubated with peroxidase-conjugatedsecondary rat IgG antibody for 30 min. The reaction was developed withABC solution Vectastain (Wertheim, Germany) and terminated by washingwith TBS. Sections were counterstained with hematoxylin-eosin,dehydrated and analyzed. FIG. 11.8 demonstrates the inhibitory effect ofthe lipid-conjugates on endotoxin-induced adhesion molecule expressionin tissues of septic rats.

The results, presented in FIGS. 11.1-11.8 demonstrate the capacity ofthe Lipid-conjugates to ameliorate the endotoxin-induced mortality amongthe septic rats (FIG. 11.2); reduced the blood level of the cytokinesTNFα and IL-6 when induced by LPS given either I.P. (FIG. 11.3), or I.V.(FIG. 11.4), and by LPS+LTA (FIG. 11.5); suppress the mRNA expression ofTNFα, IL-1 and IL-6 (FIG. 11.6), and of secretory phospholipase A₂(sPLA₂-IIA) and the inducible nitric oxide synthase (iNOS) in the lungand kidney of the septic rats (FIG. 11.7); and suppress the expressionof the adhesion molecule ICAM-1 in lung and kidney of the septic rats(FIG. 11.8). Additional support for the Lipid-conjugates to protect frombacterial toxicity is provided in Example 12 below. These resultsclearly demonstrate the therapeutic capacity of the Lipid-conjugates inthe treatment of sepsis.

EXAMPLE 12 Lung Injury/Acute Respiratory Distress Syndrome (ARDS)

In acute respiratory distress syndrome (ARDS), which is usually inducedby bacterial endotoxins (LPS, LTA), a high production of injuriousmediators, particularly neutrophil-attracting chemokines, and cytokines,are produced by the lung microvascular endothelial cells (LMVEC). Todemonstrate the ability of the Lipid-conjugates to control theproduction of these injurious agents, LMVEC were treated with LPS(gram-positive bacterial endotoxin) and LTA (gram-negative bacterialendotoxin), in the absence and presence of Lipid-conjugates, and testedfor the subsequent production of cytokines and adhesion molecules.

To this end, human lung microvascular endothelial cells (LMVEC) werepurchased from CellSystems, Remagen, Germany at passage 4. The cellswere seeded in a density of 5000 cells^(−cm2) in T25 flasks andmaintained according to the manufacturer's specification in EGM-MV.Characterization of the LMVEC was performed on the basis of a positivestaining for uptake of acetylated LDL, Factor VIII related antigen andPECAM (CD31) expression as well as negative staining for alpha smoothmuscle actin. In each experiment the viability of LPS- andLTA-stimulated or HyPE-treated LMVEC was tested by trypan blueexclusion. The production and mRNA expression of cytokines and adhesionmolecules were determined as described in Example 11 above.

The production of the chemokines ENA-78, Gro-α and IL-8, secreted intothe culture medium of stimulated LMVEC, was measured by ELISAs accordingto the manufacturer's instructions.

For RNA isolation and Polymerase Chain Reaction by RT-PCR, confluentLMVEC were stimulated with medium as control or with LPS (1 μg^(−ml)) orLTA (10 μg^(−ml)) in the presence or absence of HyPE (10 μM). Total RNAwas isolated using Trizol-Reagent according to the manufacturer'sinstructions. Each RNA preparation was subjected to DNAse digestion toremove possible contaminations of genomic DNA. 1 μg of total RNA wasreverse transcribed using SuperScript™ II Preamplification Systemaccording to the manufacturer's instructions. Amplification of 0.5 μl ofcDNA was performed in a total volume of 25 μl containing 19.6 pmol ofeach chemokine primer, 5 mM of dNTPs, 2.5 U Taq Polymerase, 10 mM TrisHCl, 7.5 mM KCl, 1.5 mM MgCl₂. PCR reactions were initiated at 94° C.for 3 min, followed by 30 cycles of amplification, each consisting of94° C. for 1 min, 58° C. for 1 min, 72° C. for 2 min. At the end of theamplification cycles the products were incubated for 10 min at 72° C.Control samples were constructed either by omitting cDNA synthesis orwithout addition of cDNA. PCR products were separated on a 1% agarosegel. Real-time PCR: 500 ng of total RNA of each sample was in additionreverse-transcribed into cDNA for Real-time PCR analysis using 1stStrand cDNA Synthesis Kit according to the manufacturer's instructions(Roche). cDNA was diluted in 20 μl DEPC-treated water. DNA standardswere generated by PCR amplification of gene products, purification andquantification by spectrophotometry. Real time PCR of cDNA specimens andDNA standards were performed in a total volume of 25 μl in the presenceof 2 μl Light cycler-FastStart DNA Master SYBR GreenI reaction mix, 0.5μM of gen-specific primers and 4 mM MgCl₂. Standard curves weregenerated for all chemokines. PCR efficiency was assessed from theslopes of the standard curves and was found to be between 90% and 100%.Concentration of chemokine cDNA was calculated by linear regressionanalysis of all standard curves and was corrected for an equalexpression of GAPDH. At least five reproducible experiments wereperformed.

Adhesion molecules ICAM-1 and p-selectin were determined byfluorescence-activated cell sorter (FACS); Confluent LMVEC werestimulated with medium as control or with LPS (1 μg^(−ml)) or LTA (10μg^(−ml)) in the presence or absence of HyPE (10 μM). Thereafter cellswere harvested by T/E, extensively washed and monoclonal antibodiesdirected against the endothelial adhesion molecules ICAM-1 andP-selectin in dilutions of 1:20 were added for 30 min at 4° C. Inaddition unstimulated or stimulated cells were harvested as describedand preincubated for 20 min with HyPE (10 μM) and monoclonal antibodiesagainst TLR4. Cells were washed and incubated with an anti-mouseF(ab′)2, FITC conjugated secondary antibody. After washing cells wereanalyzed by FACS-scan.

Expression of NFκB was determined by Electrophorese mobility shift assay(EMSA); Confluent LMVEC were preincubated overnight in basal mediumcontaining 0.01% BSA. Thereafter they were stimulated or not fordifferent time periods with LPS, IL-1 or TNF-α in the presence orabsence of HyPE, and respective nuclear extracts were prepared.Oligonucleotides containing a NFkB consensus sequence (5′-AGT TGA GGGGAC TTT CCC AGG C-3′) were labeled to a specific activity >5×107cpm^(−μg) DNA. NF-kB-binding was performed in 10 mM HEPES, (pH=7.5), 0.5mM EDTA, 70 mM KCl, 2 mM DTT, 2% glycerol, 0.025% NP-40, 4% Ficoll, 0.1M PMSF, 1 mg^(−ml) BSA and 0.1 μg^(−μl) poly di/dc in a total volume of20 μl. Nuclear extracts (10 μg) were incubated for 30 minutes at roomtemperature in the presence of 1 ng labeled oligonucleotide. DNA-proteincomplexes were resolved on 5% non-denaturating polyacrylamide gelselectrophoresed in low ionic strength buffer and visualized byautoradiography. Specificity of shifted bands was demonstrated by addinga cold NFkB consensus sequence or by supershift using anti-p65antibodies.

Experiment 12.1 demonstrates that the Lipid-conjugates are effective insuppressing the endotoxin-induced production and RNA expression of thechemokines IL-8, ENA-78 and Gro-α and their mRNA expression, as shown inFIGS. 12.1, 12.2 and 12.3.

Experiment 12.2 demonstrates that the Lipid-conjugates are effective insuppressing the expression of the adhesion molecules ICAM-1 andE-selectin (FIG. 12.4).

Experiment 12.3 demonstrates that Lipid-conjugates are effective insuppressing the expression of NFκB, the transcription factor that isenhanced in endotoxin-induced injurious states (FIG. 12.5).

Together with the experiments of Example 11, these results furtherdemonstrate the therapeutic capacity of the Lipid-conjugates in thetreatment of ARDS and lung injuries, as well as other disease that sharecommon mechanisms, such as peritonitis, kidney failure, organtransplantation and the like.

EXAMPLE 13 Transplant organ rejection, alloimmune, and autoimmunedisease

The Lipid-conjugates are effective therapy in the treatment ofautoimmune and alloimmune disease, including treatment for tissuetransplantation. This is demonstrated in experiments 13.1-13.5 below.Alloimmune disease includes tissue damage due to the immune responsewhen tissue, including blood products and whole organs, is transplantedfrom a donor to a recipient. This response is frequently directedagainst blood vessel tissue. Autoimmune disease may involve any organvia immune mediated destruction directly of the parenchyma or throughthe organ's vasculature. Two events dominant in either disease processare the proliferation of lymphocytes and immunological responsesinvolving the MHC group of antigens. Commonly accepted demonstrations ofthe immunosuppressive effect of a drug are the ability to inhibitlymphocyte proliferation and the ability to inhibit the expression ofthe MHC group of antigens.

Experiments 13.1-13.2 demonstrate that the Lipid-conjugates suppress theexpression of the human MHC antigen group, both at the basal level, andupon exposure to a stimulatory agent.

Experiment 13.1: Human proximal tubular endothelial cells (PTEC)cultured to confluency in human endothelial growth medium were incubatedin control or IFN-γ supplemented medium (10 ng/ml) in the absence orpresence of HYPE (10 μM) for the indicated time. The cells were washedand then mobilized by trypsinization and incubated for 30 min withspecific antibodies fluorescently labeled with FITC. The expression ofMHC-1, MHC-2, and ICAM was determined by FACS and expressed as themedian of the respective cell-associated fluorescence intensity (Table13.1). TABLE 13.1 Effect of HYPE on Basal and IFN-γ-Induced Expressionof MHC Class I, Class II, and ICAM-1 in PTEC Basal expressionIFN-γ-induced expression^(a) −HYPE +HYPE^(b) P −HYPE +HYPE^(b) p MHC 75± 12^(c) 11 ± 4 <0.01 1040 ± 72  87 ± 16 <0.01 Class I MHC —^(ud) —^(ud)94 ± 8 6.6 ± 8   <0.01 Class II ICAM- 15 ± 3    6.5 ± 2  <0.05 38 ± 5 7± 5 <0.01 1^(a)PTEC were stimulated with 100 ng/ml of IFN-γ for 72 h.^(b)HYPE was used in a concentration of 1 mg/ml.^(c)Results are expressed as mean fluorescence intensity ± SD using datafrom three independent experiments.^(ud)Undetectable.

Experiment 13.2: Lipid-conjugates inhibit the MHC-I expression byendothelial cells: Human umbilical vein endothelial cells were incubatedfor 72 h in culture medium (control) or stimulated with INFγ, in theabsence or presence of HYPE. The same procedure as in the previous Tablewas applied. The expression of MHC-1 was determined by FACS andexpressed as the median of the respective cell-associated fluorescenceintensity (FIG. 13-1).

Experiment 13.3: To demonstrate that the Lipid-conjugates inhibit theability of lymphocytes from both healthy and diseased animals toproliferate in response to various stimulatory agents, pooled lymph nodecells (LNC) were prepared from four mice. The in vitro response of LNCwas assayed in triplicate in a 96 well plate. LNC 2.5×10⁵ were added toeach well, together with Concanavalin A (Con A, 1 μg/ml),proteolipoprotein (PLP, 10 μg/ml), and LPS (50 μg/ml) in the presence orabsence of CMPE (10 μM) for 96 h. During the final 18 h, 1 μCi/well³[H]thymidine was added to each well, after which the plate washarvested onto a glass fiber filter, and counted in scintillation fluid.FIG. 13.2 demonstrates the ability of the Lipid-conjugates to inhibitthe proliferation of activated T-cells.

Experiment 13.4: Modulation of T-lymphocyte proliferation in response tomixed lymphocyte reaction (MLR): Because the proliferation ofallospecific T lymphocytes requires the recognition of MHC class II, itwas investigated whether the Lipid-conjugates can influence T-cellactivation stimulated in mixed lymphocyte reaction (MLR, with dendriticcells). To this end, peripheral blood leukocytes (PBL) were isolatedfrom two HLA-incompatible individuals by Ficoll. PBL from one individualwere used to isolate dendritic cells by cultivating adherent mononuclearcells in the presence of granulocyte macrophage-colony-stimulatingfactor (GM-CSF) (800 U/ml) and IL-4 (1000 U/ml) (both from R&D Systems)for 7 days. On day 7, the cultures were stimulated for 3 days in thepresence of IL-4 and GM-CSF with a cocktail of IL-1 (10 ng/ml), IL-6(1000 U/ml), PGE2 (1 g/ml), and tumor necrosis factor (TNF-α, 10 ng/ml).Thereafter dendritic cells were irradiated (30 Gy) and used asstimulators. T cells from an HLA-incompatible individual were purifiedby negative selection using minimacs and used as responder in mixedlymphocyte reaction (MLR). MLR reactions were set up in differentstimulator:responder ratios for 3, 5, or 8 days in the presence orabsence of HYPE. Proliferation was measured by means of BrdUincorporation using a cell-based ELISA system (BrdU labeling anddetection kit III, Roche, Mannheim, Germany) according to themanufacturer's instructions.

FIG. 13.3 shows that lymphocyte proliferation was strongly impaired byHYPE in MLR. A significant inhibition was still observed when dendriticcells were preincubated for 24 hr with 1 mg/ml of HYPE and used asstimulator cells in MLR in the absence of HYPE (*P<0.01; **P<0.05, bystatistical analysis performed by ANOVA with Bonferroni adjustment formultiple testing).

Experiment 13.5: Cytokine production by T-lymphocytes subjected to MLR:PBL were isolated as described and cultured in MLR, in the presence orabsence of HYPE (1 mg/mil). Culture supernatants from MLR were collectedon day 5 and were analyzed for the production of IFN-, IL-2, IL-4,IL-10, and IL-12 by ELISA (all from R&D Systems) performed according tothe manufacturer's instructions. The Lipid-conjugate effect isdemonstrated in Table 13.2: TABLE 13.2 HyPE inhibits cytokine productionby MLR-stimulated lymphocytes Medium +HyPE (10 μM)  IL-2 (pg/ml) 570 ±20  73 ± 12* IFN-γ (pg/ml) 2250 ± 243 500 ± 63* IL-10 (pg/ml) 39 ± 4  8± 2***P < 0.01,**P < 0.05.Each datum is mean ± SD for 3 experiments.

In addition to the immune response, transplant rejection is facilitatedby ischemia/reperfusion injury and damage by oxygen radicals. Datapresented in the previous examples above demonstrate that theLipid-conjugates prevents white cell activation induced byischemia/reperfusion (Example 7.4), and are effective as anti-oxidanttherapy (Example 9). Taken together, the data presented here demonstratethat the Lipid-conjugates provide effective therapy for prevention oftransplant rejection.

EXAMPLE 14 Viral Infection

The Lipid-conjugates are effective in the prophylaxis and treatment ofviral infection, particularly the infections due to the humanimmunodeficiency virus (HIV). This is demonstrated in Experiment 14.1below The process of viral infection comprises stages in which freeviral particles are able to enter host cells and produce signs ofillness. A commonly accepted assay for anti-viral activity of a drug isto incubate a preparation of the viral agent in the presence of thedrug, followed by testing for viral infection in a human cell line.

Experiment 14.1: To demonstrate that the Lipid-conjugates are capable ofpreventing HIV infection of target cells, whole blood units were mixedwith HIV and a Lipid-conjugate (50 μM HEPPE, 30 μM HYPE) for 30 min. Thecells were then spun and the supernatant was examined for HIVinfectivity on HT4-1022 cells as described by Margolis-Nunno et al(Transfusion, 36, 743-750, 1996). FIG. 14.1 demonstrates the ability ofLipid-conjugates to prevent HIV infection of cells.

Experiment 14.2: Inhibition of HIV-1_(IIIB) Infection

Table 14.2 demonstrates the capacity of the Lipid-conjugates to inhibitHIV replication, as expressed by the production of the nucleocapsid p24antigen, which is produced in the host cell upon its infection by HIVvirus: ³¹MT-2 cells (10⁴) in 96-well plates were infected with HIV-1 (adose sufficient to accomplish a multiplicity of infection of 0.0045) in200 μl of RPMI 1640 medium supplemented with 10% (v/v) fetal bovineserum (FBS), in the absence (control) and presence of the indicatedLipid-conjugate. After 1 h and 24 h, respectively, half of the culturemedium was changed and replaced by fresh medium (with/withoutLipid-conjugate). On the fourth day after incubation at 37° C., 100 μlof culture supernatants were collected from each well and an equalvolume of fresh medium was added to the wells. The collectedsupernatants were mixed with an equal volume of 5% (v/v) Triton X-100and assayed for p24 antigen using an ELISA kit from Coulter Immunology(Hialeah, FL). TABLE 14.1 Inhibition of p24 production Compounds IC₅₀ (M± SD) μg/ml IC₉₀ (M ± SD) μg/ml HyPE 207.0 ± 18.0 384.3 ± 79.3 CSAPE 72.5 ± 8.0 106.0 ± 10.3 HepPE  10.0 ± 2.3  19.3 ± 4.5 HemPE 375.8 ±119.5 >500 HyDMPE 118.0 ± 16.8 296.3 ± 104.0

Experiment 14.3: Inhibition of fusion between HIV-infected withHIV-uninfected cells: The anti-HIV-1 activity of the Lipid-conjugateswas evaluated by measuring the inhibition of fusion between HIV-1infected and uninfected cells.

In this assay, HIV-1_(IIIB).infected H9 cells were labeled with BCECF(2′,7′-bis(2-carboxyethyl)-5-6-carboxyfluorescein-acetoxymethyl-ester,Molecular Probes, Eugene, Oreg.) according to the manufacturer'sinstructions. BCECF-labeled H9/HIV-1 IIIB cells (10⁴) were mixed with1×10⁵ uninfected MT-2 cells After incubation in a 96-well plate at 37°C. for 2 h, the fused and unfused labeled cells were counted under aninverted fluorescence microscope at ×160 magnification. At least 200BCECF-labeled cells were counted and the proportion of fused cells wasdetermined. These tests were carried out in the presence and absence ofgraded quantities of the tested Lipid-conjugates, as shown in Table14.2. TABLE 14.2 Inhibition of cell fusion between HIV-infected anduninfected cells. Compounds IC₅₀ (M ± SD) μg/ml IC₉₀ (M ± SD) μg/mlHYPE >500 >500 CSAPE >500 >500 HepPE 7.9 ± 1.3 15.3 ± 3.9HemPE >500 >500 HyDMPE 122.8 ± 14.8  219.8 ± 10.6

These experiments demonstrate that administration of Lipid-conjugates iseffective therapy in the treatment of viral infection, particularly HIV,and useful in the eradication of viral particles from contaminatedmaterials, including blood products.

EXAMPLE 15 Treatment of Conjunctivitis

The Lipid-conjugates are effective in treatment of hypersensitivityconjunctivitis induced by the delayed-type hypersensitivity immuneresponse. This is demonstrated in Experiment 15.1 below.

Experiment 15.1: Guinea pigs were sensitized by two I.P. injections (oneweek between injections) with 10 mg ovalbumin dissolved in 0.5 ml PBS,supplemented with Freunds adjuvant. Three weeks after the originalsensitization the first challenge was performed by dripping 5 mgovalbumin dissolved in 25 ml PBS (FIG. 15.1) and repeated challengeswere performed 3,4,5, and 6 days after the first challenge (FIG. 15.2).For treatment the drug (CMPE), suspended in PBS was dripped into theright eye of each animal on days 3,4,5, and 6 after the first challenge.Clinical evaluation of corneal opacity was done on days 5 and 6.Ophthalmic levels of LTB4 and PGE₂ were determined by ELISA (FIG. 15.3).For comparison, the effect of steroid treatment was evaluated inparallel. These results demonstrate the Lipid-conjugate ability toameliorate allergen-induced conjunctivitis.

EXAMPLE 16 Treatment of Chlamydia Infection

The Lipid-conjugates are effective in the prophylaxis and treatment ofinfection with intracellular bacterial parasites, particularlyinfections due to chlaniydial species This is demonstrated inExperiments 16.1-16.2 below.

Experiment 16.1: Human cervical adenocarcinoma cell line, HeLa 229(ATCC, Manassas, Calif.), were cultured and incubated with the PLconjugates (20 micromolar) for 30 min, then incubated with Chlamydiapsittaci (guinea pig inclusion conjunctivitis servovar) for 24 hr.Infected cells were detected by cytofluorometry (FACS) usingFITC-conjugated anti-Chlamydia antibody (FIG. 16.1A).

FIG. 16.1B depicts the dose response of the Lipid-conjugates inhibitoryeffect on infection of HeLa cells by Chlamydia: HeLa cells were treatedwith the Lipid-conjugates at the indicated concentration, and infectedwith Chlamydia as above.

Experiment 16.2: Inhibition of Chlamydia-induced cell apoptosis: HeLacells were treated with Lipid-conjugates and infected with Chlamydiapsittaci as in Experiment 16.1. For determination of apoptosis,detergent-permeabilized cells were stained with propidium iodide, andtheir fluorescence was measured by cytofluorometry (FIG. 16.2).

Additional support is provided in Examples 11-12, showing that theLipid-conjugates protect from gram-negative and gram-positiveendotoxins. Taken together, the data presented here demonstrate theLipid-conjugate capacity to ameliorate bacterial toxicity.

EXAMPLE 17 Toxicity Tests

Experiment 17: The following compounds were tested: HyPE, CMPE, CSAPEand HepPE. The compounds were injected IP at one dose of 1000, 500 or200 mg/Kg body weight. Toxicity was evaluated after one week, bymortality, body weight, hematocrit, blood count (red and white cells),and visual examination of internal organs after sacrifice. These werecompared to control, untreated mice. Each dose was applied to a group ofthree mice. No significant change in the above criteria was induced bytreatment with these compounds, except for the HepPE, which inducedhemorrhage.

The non-toxicity of the Lipid conjugates is demonstrated in Table 17.1and Table 17.2, depicting the results obtained for HyPE in acute (17.1)and long-term (17.2) toxicity tests. TABLE 17.1 Acute toxicity Dose ofHyPE Hemato- (mg/kg RBC × WBC × crit body weight) Body weight (g) 10⁶10³ %    0.0 21.9 ± 0.2 22.6 ± 0.3 10.7 ± 0.4 9.3 ± 0.3 45.0 ± 0.5(control)  250 22.1 ± 0.4 23.1 ± 0.6 11.4 ± 0.1 7.7 ± 0.2 43.3 ± 0.7 500 21.4 ± 0.3 22.3 ± 0.4 11.5 ± 0.3 8.1 ± 1.3 44.7 ± 2.3 1000 21.7 ±0.2 22.1 ± 0.2 10.9 ± 0.4 7.4 ± 0.6 40.3 ± 0.7RBC = red blood cells.WBC = white blood cells.Each datum is mean ± SEM.

For long-term toxicity test of HyPE, a group of 6 mice received a doseof 100 mg HyPE/Kg body weight, injected IP 3 times a week for 30 weeks(total of 180 mg to a mouse of 20 g). Toxicity was evaluated as forTable 17.1. No mortality, and no significant change in the abovecriteria was induced by this treatment, compared to normal untreatedmice (see Table 17.1), as depicted in Table 17.2. TABLE 17.2 Results atweek 30: Body RBC WBC weight (g) ×10⁶ ×10³ Hematocrit % Control(untreated) 39.5 ± 3.1 10.9 ± 0.8 9.3 ± 0.6 45.0 ± 0.8 ratsHyPE-injected rats 39.0 ± 2.7 11.7 ± 0.7 8.1 ± 15  43.4 ± 4.9

EXAMPLE 18 Synthesis Procedures

The procedures below are examples for synthesis of specific variants ofthe lipid-conjugates, and can be modified according to the desirablecompositions (e.g, changing the molar ratio between thelipid/phospholipid and the GAG, or the GAG size).

I. HyPE=phosphatidyl-ethanolamine (PE)-linked hyaluronic acid.

A. Truncating hyaluronic acid (HA):

Dissolve 20 g of HA in 12 L water, add 200 mg FeSO₄.7H₂O dissolved in 20ml water, add 400 ml H₂O₂ (30%), stir for 1.5 h. Filter through 30 kDFiltron, Lyophilize. Yield: 16 g truncated HA.

B. Conjugation with PE (adjusted for 1 g):

Prepare:

-   -   1. 10 g HA dissolved in 500 ml MES buffer, 0.1 M, pH=6.5    -   2. 1.0 g PE dissolved in 500 ml t-BuOH with 100 ml H₂O.        Mix the two solutions, add 1 g HOBT and 10 g EDC. Sonicate the        mixture in an ultrasonic bath for 3 h. Remove access free PE        (and EDC and HOBT) by extraction into organic phase (by addition        of chloroform and methanol to obtain a ratio of C/M/H₂O:1/1/1).        Separate the aqueous phase by a separation funnel. Repeat this        step twice. For final cleaning from reagents, filter through a        Filtron membrane (30 kD), and lyophilize.

Yield: about 8 g.

II. CSAPE=PE-linked chondroitin sulfate A (CSA):

Prepare:

-   -   1.10 g CSA dissolved in 1.2 L MES buffer, 0.1 M, pH=6.5    -   2. 1 g PE dissolved in 120 ml chloroform/methanol: 1/1. Add 15        ml of a detergent (DDAB).        Mix 1 with 2, while stirring, add 1 g HOBT and 10 g EDC,        continue stirring thoroughly for a day at least. Remove access        free PE (and EDC and HOBT) by extraction into organic phase (by        addition of chloroform and methanol to obtain a ratio of        Chloroform/MeOH/EtOH/H₂O: 1/1/0.75/1). Separate the aqueous        phase by a separation funnel. Repeat this step twice. Filter        through a Filtron membrane (30 kD), and lyophilize. To remove        DDAB traces, dissolve 1 g of dry product in 100 ml water and 100        ml MeOH, and clean by ion exchanger using IR120 resin. Dialyse        (to remove MeOH) and lyophilize.

Yield: about 8 g.

Unexpected results showed that the sonication applied in the HyPEsynthesis, is an better substitute for the detergent in mixing theaqueous and lipid phases. Using sonication techniques simplifies thesynthesis and improves the purification of the product.

REFERENCES

-   1. Krimsky et al., Journal of Basic and Clinical Physiology and    Pharmacology 11:143-153, 2000.-   2. Krimsky et al., American Journal of Physiology 285:G586-G592,    2003.-   3. Murthy et al. Dig Dis Sci, 38, 1722, 1993.-   4. Okayasu et al., Gastoenterology, 98, 694, 1990.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed herein above and that numerous modifications, all of whichfall within the scope of the present invention, exist. Rather, the scopeof the invention is defined by the claims which follow:

1. A compound represented by the structure of the general formula (A):

wherein L is a lipid or a phospholipid; Z is either nothing,ethanolamine, serine, inositol, choline, or glycerol; Y is eithernothing or a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer, or polymer, whereinx is a glycosaminoglycan; and n is a number from 1 to 1000; wherein anybond between L, Z, Y and X is either an amide or an esteric bond.
 2. Acompound according to claim 1, represented by the structure of thegeneral formula (I):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Y is eithernothing or a spacer group ranging in length from 2 to 30 atoms; X iseither a physiologically acceptable monomer, dimer, oligomer or aphysiologically acceptable polymer, wherein X is a glycosaminoglycan;and n is a number from 1 to 1,000; wherein if Y is nothing thephosphatidylethanolamine is directly linked to X via an amide bond andif Y is a spacer, said spacer is directly linked to X via an amide or anesteric bond and to said phosphatidylethanolamine via an amide bond. 3.A compound according to claim 1, represented by the structure of thegeneral formula (II):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Y is eithernothing or a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer or polymer wherein xis a glycosaminoglycan; and n is a number from 1 to 1000; wherein if Yis nothing the phosphatidylserine is directly linked to X via an amidebond and if Y is a spacer, said spacer is directly linked to X via anamide or an esteric bond and to said phosphatidylserine via an amidebond.
 4. A compound according to claim 1, represented by the structureof the general formula (III):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, inositol, choline, or glycerol; Y is either nothing or a spacergroup ranging in length from 2 to 30 atoms; X is a physiologicallyacceptable monomer, dimer, oligomer, or polymer, wherein x is aglycosaminoglycan; and n is a number from 1 to 1000; wherein any bondbetween the phosphatidyl, Z, Y and X is either an amide or an estericbond.
 5. A compound according to claim 1, represented by the structureof the general formula (IV):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, inositol, choline, or glycerol; Y is either nothing or a spacergroup ranging in length from 2 to 30 atoms; X is a physiologicallyacceptable monomer, dimer, oligomer, or polymer, wherein x is aglycosaminoglycan; and n is a number from 1 to 1000; wherein any bondbetween the phospholipid, Z, Y and X is either an amide or an estericbond.
 6. A compound according to claim 1, represented by the structureof the general formula (V):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, inositol, choline, or glycerol; Y is eithernothing or a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer, or polymer, whereinx is a glycosaminoglycan; and n is a number from 1 to 1000; wherein anybond between the phospholipid, Z, Y and X is either an amide or anesteric bond.
 7. A compound according to claim 1, represented by thestructure of the general formula (VI):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, inositol, choline, or glycerol; Y is either nothing or a spacergroup ranging in length from 2 to 30 atoms; X is a physiologicallyacceptable monomer, dimer, oligomer, or polymer, wherein x is aglycosaminoglycan; and n is a number from 1 to 1000; wherein any bondbetween the phospholipid, Z, Y and X is either an amide or an estericbond.
 8. A compound according to claim 1, represented by the structureof the general formula (VII):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, inositol, choline, or glycerol; Y is eithernothing or a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer, or polymer, whereinx is a glycosaminoglycan; and n is a number from 1 to 1000; wherein anybond between the phospholipid, Z, Y and X is either an amide or anesteric bond.
 9. A compound according to claim 1, represented by thestructure of the general formula (VIII):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, ethanolamine, serine, inositol, choline, orglycerol; Y is either nothing or a spacer group ranging in length from 2to 30 atoms; X is a physiologically acceptable monomer, dimer, oligomer,or polymer, wherein x is a glycosaminoglycan; and n is a number from 1to 1000; wherein any bond between the phospholipid, Z, Y and X is eitheran amide or an esteric bond.
 10. A compound according to claim 1,represented by the structure of the general formula (IX):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, ethanolamine, serine, inositol, choline, orglycerol; Y is either nothing or a spacer group ranging in length from 2to 30 atoms; X is a physiologically acceptable monomer, dimer, oligomer,or polymer, wherein x is a glycosaminoglycan; and n is a number from 1to 1000; wherein any bond between the phospholipid, Z, Y and X is eitheran amide or an esteric bond.
 11. A compound according to claim 1,represented by the structure of the general formula (X):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, ethanolamine, serine, inositol, choline, or glycerol; Y iseither nothing or a spacer group ranging in length from 2 to 30 atoms; Xis a physiologically acceptable monomer, dimer, oligomer, or polymer,wherein x is a glycosaminoglycan; and n is a number from 1 to 1000;wherein any bond between the ceramide phosphoryl, Z, Y and X is eitheran amide or an esteric bond.
 12. A compound according to claim 1,represented by the structure of the general formula (XI):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is nothing; Y is either nothing or a spacer group ranging inlength from 2 to 30 atoms; X is a physiologically acceptable monomer,dimer, oligomer or polymer, wherein x is a glycosaminoglycan; and n is anumber from 1 to 1000; wherein if Y is nothing the sphingosyl isdirectly linked to X via an amide bond and if Y is a spacer, said spaceris directly linked to X and to said sphingosyl via an amide bond and toX via an amide or an esteric bond.
 13. A compound according to claim 1,represented by the structure of the general formula (XII):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, ethanolamine, serine, inositol, choline, or glycerol; Y iseither nothing or a spacer group ranging in length from 2 to 30 atoms; Xis a physiologically acceptable monomer, dimer, oligomer or polymer,wherein x is a glycosaminoglycan; and n is a number from 1 to 1000;wherein any bond between the ceramide, Z, Y and X is either an amide oran esteric bond.
 14. A compound according to claim 1, represented by thestructure of the general formula (XIII):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, choline, phosphate, inositol, or glycerol; Y is either nothingor a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer or polymer, whereinx is a glycosaminoglycan; and n is a number from 1 to 1000; wherein anybond between the diglyceryl, Z, Y and X is either an amide or an estericbond.
 15. A compound according to claim 1, represented by the structureof the general formula (XIV):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, choline, phosphate, inositol, or glycerol; Y is either nothingor a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer or polymer, whereinx is a glycosaminoglycan; and n is a number from 1 to 1000; wherein anybond between the glycerolipid, Z, Y and X is either an amide or anesteric bond.
 16. A compound according to claim 1, represented by thestructure of the general formula (XV):

wherein R₁ is a linear, saturated, mono-unsaturated, orpoly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, choline, phosphate, inositol, or glycerol; Yis either nothing or a spacer group ranging in length from 2 to 30atoms; X is a physiologically acceptable monomer, dimer, oligomer orpolymer, wherein x is a glycosaminoglycan; and n is a number from 1 to1000; wherein any bond between the glycerolipid, Z, Y and X is either anamide or an esteric bond.
 17. A compound according to claim 1,represented by the structure of the general formula (XVI):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, choline, phosphate, inositol, or glycerol; Y is either nothingor a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer or polymer, whereinx is a glycosaminoglycan; and n is a number from 1 to 1000; wherein anybond between said lipid, Z, Y and X is either an amide or an estericbond.
 18. A compound according to claim 1, represented by the structureof the general formula (XVII):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is a linear, saturated, mono-unsaturated, or poly-unsaturated,alkyl chain ranging in length from 2 to 30 carbon atoms; Z is eithernothing, choline, phosphate, inositol, or glycerol; Y is either nothingor a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer or polymer, whereinx is a glycosaminoglycan; and n is a number from 1 to 1000; wherein anybond between the lipid, Z, Y and X is either an amide or an estericbond.
 19. A compound according to claim 1, represented by the structureof the general formula (XVIII):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, choline, phosphate, inositol, or glycerol; Yis either nothing or a spacer group ranging in length from 2 to 30atoms; X is a physiologically acceptable monomer, dimer, oligomer orpolymer, wherein x is a glycosaminoglycan; and n is a number from 1 to1000; wherein any bond between the lipid, Z, Y and X is either an amideor an esteric bond.
 20. A compound according to claim 1, represented bythe structure of the general formula (XIX):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, choline, phosphate, inositol, or glycerol; Yis either nothing or a spacer group ranging in length from 2 to 30atoms; X is a physiologically acceptable monomer, dimer, oligomer orpolymer, wherein x is a glycosaminoglycan; and n is a number from 1 to1000; wherein any bond between the lipid, Z, Y and X is either an amideor an esteric bond.
 21. A compound according to claim 1, represented bythe structure of the general formula (XX):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, choline, phosphate, inositol, or glycerol; Yis either nothing or a spacer group ranging in length from 2 to 30atoms; X is a physiologically acceptable monomer, dimer, oligomer orpolymer, wherein x is a glycosaminoglycan; and n is a number from 1 to1000; wherein any bond between the lipid, Z, Y and X is either an amideor an esteric bond.
 22. A compound according to claim 1, represented bythe structure of the general formula (XXI):

wherein R₁ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; R₂ is either hydrogen or a linear, saturated, mono-unsaturated,or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbonatoms; Z is either nothing, choline, phosphate, inositol, or glycerol; Yis either nothing or a spacer group ranging in length from 2 to 30atoms; X is a physiologically acceptable monomer, dimer, oligomer orpolymer, wherein x is a glycosaminoglycan; and n is a number from 1 to1000; wherein any bond between the lipid, Z, Y and X is either an amideor an esteric bond.
 23. The compound according to claim 1, wherein theglycosaminoglycan is hyaluronic acid, heparin, heparan sulfate,chondrotin sulfate, keratin, keratan sulfate, dermatan sulfate or aderivative thereof.
 24. The compound according to claim 23, wherein theglycosaminoglycan is di- and trisaccharide unit monomers ofglycosaminoglycans.
 25. The compound according to claim 24, wherein thechondroitin sulfate is chondroitin-6-sulfate, chondroitin-4-sulfate or aderivative thereof.
 26. The compound according to claim 1, wherein thesugar rings of said glycosaminoglycan are intact.
 27. A process for thepreparation of a compound represented by the structure of the generalformula (A):

wherein L is a lipid or a phospholipid; Z is either nothing,ethanolamine, serine, inositol, choline, or glycerol; Y is eithernothing or a spacer group ranging in length from 2 to 30 atoms; X is aphysiologically acceptable monomer, dimer, oligomer, or polymer, whereinX is a glycosaminoglycan; and n is a number from 1 to 1000; wherein anybond between L, Z, Y and X is either an amide or an esteric bond,comprising the steps of: conjugating L to Z; conjugating Z to Y;conjugating Y to X; wherein if Z is nothing, L is conjugated directly toY, if Y is nothing, Z is conjugated directly to X, and if Y and Z arenothing, L is conjugated directly to X, thereby preparing a compoundrepresented by the structure of the general formula (A).
 28. The processaccording to claim 27, wherein said conjugating is performed byeliminating a water molecule, thereby forming amide or esteric bonds.29. The process according to claim 27, wherein said conjugating isperformed in the presence of a detergent.
 30. The process according toclaim 27, wherein said conjugating is induced by ultrasonic radiation.31. The process according to claim 27, wherein the sugar rings of saidglycosaminoglycan remain closed during all steps.